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Sample records for 3t3 cells transfected

  1. Resistance to anticancer drugs in NIH3T3 cells transfected with c-myc and/or c-H-ras genes.

    PubMed Central

    Niimi, S.; Nakagawa, K.; Yokota, J.; Tsunokawa, Y.; Nishio, K.; Terashima, Y.; Shibuya, M.; Terada, M.; Saijo, N.

    1991-01-01

    NIH3T3 cells transfected with c-H-ras and/or c-myc genes were examined for differences in drug sensitivity. The five transfectants used were N8, NIH3T3-nm-1, pT22-3-nm-2, pP1-4 and pT22-3. They were transfected with pKOneo alone, pKOneo and c-myc, pKOneo and c-myc plus activated c-H-ras, normal c-H-ras and activated c-H-ras genes, respectively. The IC50s of cisplatin, 4-hydroperoxycyclophosphamide, adriamycin, melphalan, and CPT-11 were significantly higher for NIH3T3-nm-1 abd pT22-3-nm-2 than for the parental NIH3T3 and N8 cells. Transfection with normal and activated C-H-ras oncogenes only led to increases in the IC50s of alkylating agents. There was no significant difference between the IC50s of N8 and those of NIH3T3 parental cells to any of these anticancer agents. These results strongly suggest that the expression of the c-myc gene plays a role in the acquisition of drug resistance. The c-myc gene may therefore provide us with an important clue in determining the mechanism of drug resistance. Images Figure 1 Figure 2 Figure 3 PMID:1997100

  2. Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells

    SciTech Connect

    Sutherland, B.M.; Bennett, P.B.; Freeman, A.G.; Moore, S.P.; Strickland, P.T.

    1985-04-01

    Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.

  3. [Envelope protein of Jaagsiekte sheep retrovious expressed in NIH3T3 cells promotes cell proliferation].

    PubMed

    DU, Fangyuan; Chen, Dayong; Zhang, Yufei; Sun, Xiaolin; Guo, Wenqing; Liu, Shuying

    2016-09-01

    Objective To explore the influence of the exogenous Jaagsiekte sheep retrovious (exJSRV) envelope protein (Env) on NIH3T3 cell proliferation. Methods A recombinant plasmid pcDNA4/myc-His/exJSRV- env carrying exJSRV- env gene was constructed, and then the correctness of the recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmid pcDNA4/myc-His/exJSRV- env was transiently transfected into NIH3T3 cells by Lipofectamine(TM) LTX. After the transfection of the recombinant plasmid, the expression of exJSRV- env was detected by reverse transcription PCR and Western blotting. The effect of Env on cell proliferation was investigated by CCK-8 assay and plate colony formation assay. Results The recombinant eukaryotic expression plasmid containing exJSRV- env was successfully constructed as identified by PCR, restriction enzyme identification and sequencing. After the recombinant plasmid was transiently transfected into NIH3T3 cells, reverse transcription PCR and Western blotting showed the expression of exJSRV- env , and Env promoted NIH3T3 cell proliferation significantly. Conclusion JSRV Env was expressed successfully in the NIH3T3 cells and promoted the proliferation of NIH3T3 cells. PMID:27609573

  4. Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells

    SciTech Connect

    Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao ); Wongsasant, Budsaba )

    1991-12-01

    The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

  5. Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

    PubMed Central

    Sakamuro, D; Furukawa, T; Takegami, T

    1995-01-01

    Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation. PMID:7745741

  6. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    SciTech Connect

    Yasumoto, S.; Burkhardt, A.L.; Doniger, J.; DiPaolo, J.A.

    1986-02-01

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.

  7. Olanzapine induces SREBP-1-related adipogenesis in 3T3-L1 cells.

    PubMed

    Yang, Li-Hung; Chen, Tzer-Ming; Yu, Sung-Tsai; Chen, Yen-Hui

    2007-09-01

    Olanzapine is a second-generation atypical antipsychotic drug (AAPD). Major side effects of olanzapine are weight gain and development of diabetes mellitus, which are risk factors of cardiovascular diseases. The possible causes of metabolic adverse effects are known as poor satiety and increased food intake due to blockade of receptors such as 5-HT(2C) in CNS. In this study, we examine the effect of olanzapine on peripheral adipogenesis using cultured 3T3-L1 cell model. Olanzapine increased triacylglyceride (TG) accumulation during 3T3-L1 preadipocyte differentiation to mature adipocyte phenotype. TG accumulation was accompanied by overexpression of fatty acid synthase and adiponectin that are the downstream genes of sterol regulatory element binding protein-1 (SREBP-1), one of the key transcription factors in lipid homeostasis. We further consisted that mostly SREBP-1 and at a lesser extent peroxisome proliferator-activated receptor gamma (PPAR-gamma), but not CCAAT/enhancer binding protein-alpha (C/EBP-alpha), were overexpressed and activated in 3T3-L1 adipocytes exposed to olanzapine. Furthermore, we showed that olanzapine enhanced the activity of SRE-1-containing LDLR promoter in transfected 3T3-L1 adipocytes and HepG2 cells. Taken together, olanzapine may cause body weight gain not only through influencing CNS receptors, but also affecting the peripheral adipogenesis regulated by SREBP-1. PMID:17651982

  8. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

    SciTech Connect

    Turley, E.A.; Moore, D.; Hayden, L.J.

    1987-06-02

    A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.

  9. Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells

    SciTech Connect

    Kuzumaki, N.; Ogiso, Y.; Oda, A.; Fujita, H.; Suzuki, H.; Sato, C.; Mullauer, L.

    1989-05-01

    A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due n not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

  10. Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

    PubMed

    Ukita, Mayumi; Yamaguchi, Taihiko; Ohata, Noboru; Tamura, Masato

    2016-06-01

    Sclerostin, a secreted protein encoded by the Sost gene, is produced by osteocytes and is inhibited by osteoblast differentiation and bone formation. Recently, a functional association between bone and fat tissue has been suggested, and a correlation between circulating sclerostin levels and lipid metabolism has been reported in humans. However, the effects of sclerostin on adipogenesis remain unexplored. In the present study, we examined the role of sclerostin in regulating adipocyte differentiation using 3T3-L1 preadipocytes. In these cells, sclerostin enhanced adipocyte-specific gene expression and the accumulation of lipid deposits. Sclerostin also upregulated CCAAT/enhancer binding protein β expression but not cell proliferation and caspase-3/7 activities. Sclerostin also attenuated canonical Wnt3a-inhibited adipocyte differentiation. Recently, the transcriptional modulator TAZ has been involved in the canonical Wnt signaling pathway. Sclerostin reduced TAZ-responsive transcriptional activity and TAZ-responsive gene expression. Transfection of 3T3-L1 cells with TAZ siRNA increased the lipid deposits and adipogenic gene expression. These results show that sclerostin upregulates adipocyte differentiation in 3T3-L1 cells, suggesting a possible role for the osteocyte-derived sclerostin as a regulator of fat metabolism and as a reciprocal regulator of bone and adipose tissues metabolism. J. Cell. Biochem. 117: 1419-1428, 2016. © 2015 Wiley Periodicals, Inc. PMID:26553151

  11. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  12. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

    Abbasi-Kenarsari, Hajar; Shafaghat, Farzaneh; Baradaran, Behzad; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  13. Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line

    PubMed Central

    Salehi-Lalemarzi, Hamed; Shanehbandi, Dariush; Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Baradaran, Behzad; Movassaghpour, Ali Akbar; Kazemi, Tohid

    2015-01-01

    Purpose: PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. Methods: CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. Conclusion: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins. PMID:26236664

  14. Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6).

    PubMed

    Dormady, S P; Zhang, X M; Basch, R S

    2000-10-24

    Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively gamma-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be gamma-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

  15. Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

    PubMed Central

    Shalloway, D; Johnson, P J; Freed, E O; Coulter, D; Flood, W A

    1987-01-01

    pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved. Images PMID:2446117

  16. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    SciTech Connect

    Papadopoulos, T.; Pfeifer, U. )

    1987-07-01

    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  17. Prolonged Induction Activates Cebpα Independent Adipogenesis in NIH/3T3 Cells

    PubMed Central

    Shao, Hsiao-Yun; Hsu, Hsue-Yin; Wu, Kuan-Sju; Hee, Siow-Wey; Chuang, Lee-Ming; Yeh, Jih-I

    2013-01-01

    Background 3T3-L1 cells are widely used to study adipogenesis and insulin response. Their adipogenic potential decreases with time in the culture. Expressing exogenous genes in 3T3-L1 cells can be challenging. This work tries to establish and characterize an alternative model of cultured adipocytes that is easier to work with than the 3T3-L1 cells. Methodology/Principal Findings Induced cells were identified as adipocytes based on the following three characteristics: (1) Accumulation of triglyceride droplets as demonstrated by oil red O stain. (2) Transport rate of 2-deoxyglucose increased after insulin stimulation. (3) Expression of fat specific genes such as Fabp4 (aP2), Slc2a4 (Glut4) and Pparg (PPARγ). Among the cell lines induced under different conditions in this study, only NIH/3T3 cells differentiated into adipocytes after prolonged incubation in 3T3-L1 induction medium containing 20% instead of 10% fetal bovine serum. Rosiglitazone added to the induction medium shortened the incubation period from 14 to 7 days. The PI3K/AKT pathway showed similar changes upon insulin stimulation in these two adipocytes. C/EBPα mRNA was barely detectable in NIH/3T3 adipocytes. NIH/3T3 adipocytes induced in the presence of rosiglitazone showed higher 2-deoxyglucose transport rate after insulin stimulation, expressed less Agt (angiotensinogen) and more PPARγ. Knockdown of C/EBPα using shRNA blocked 3T3-L1 but not NIH/3T3 cell differentiation. Mouse adipose tissues from various anatomical locations showed comparable levels of C/EBPα mRNA. Conclusions/Significance NIH/3T3 cells were capable of differentiating into adipocytes without genetic engineering. They were an adipocyte model that did not require the reciprocal activation between C/EBPα and PPARγ to differentiate. Future studies in the C/EBPα independent pathways leading to insulin responsiveness may reveal new targets to diabetes treatment. PMID:23326314

  18. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    SciTech Connect

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  19. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

    SciTech Connect

    Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. Black-Right-Pointing-Pointer Overexpression of ATF3 represses C/EBP{alpha} expression. Black-Right-Pointing-Pointer ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Black-Right-Pointing-Pointer ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP{alpha} transcript and repressed the activity of the 3.6-kb mouse C/EBP{alpha} promoter, demonstrating that ATF3 downregulates C/EBP{alpha} expression. Transfection studies using mutant constructs containing 5 Prime -deletions in the C/EBP{alpha} promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP{alpha} mRNA and repress the promoter activity of the C/EBP{alpha} gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP{alpha} expression. Collectively, these results demonstrate that ATF3 represses the C/EBP{alpha} gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition

  20. Effect of Biodegradable Shape-Memory Polymers on Proliferation of 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Xu, Shuo-Gui; Zhang, Peng; Zhu, Guang-Ming; Jiang, Ying-Ming

    2011-07-01

    This article evaluates the in vitro biocompatibility for biodegradable shape-memory polymers (BSMP) invented by the authors. 3T3 cells (3T3-Swiss albino GNM 9) of primary and passaged cultures were inoculated into two kinds of carriers: the BSMP carrier and the control group carrier. Viability, proliferation, and DNA synthesis (the major biocompatibility parameters), were measured and evaluated for both the BSMP and naked carrier via cell growth curve analysis, MTT colorimetry and addition of 3H-TdR to culture media. The results showed that there was no difference between the BSMP carrier and the control dish in terms of viability, proliferation, and metabolism of the 3T3 cells. Overall, the BSMP carrier provides good biocompatibility and low toxicity to cells in vitro, and could indicate future potential for this medium as a biological material for implants in vivo.

  1. CREB Activation Induces Adipogenesis in 3T3-L1 Cells

    PubMed Central

    Reusch, Jane E. B.; Colton, Lilliester A.; Klemm, Dwight J.

    2000-01-01

    Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues. PMID:10629058

  2. Viscoelastic analysis of high molecular weight, alkali-denatured DNA from mouse 3T3 cells.

    PubMed Central

    Uhlenhopp, E L

    1975-01-01

    Alkaline lysates of mouse 3T3 cells showed viscoelastic properties characteristic of very large molecules of single-stranded DNA. The viscoelastic retardation time and the sensitivity to low doses of nitrogen mustard and of X-irradiation suggest a molecular weight in excess of 10-10 daltons. Contact-inhibited cells yielded larger single strands than actively growing cells. PMID:235335

  3. Expression of Nanog gene promotes NIH3T3 cell proliferation

    SciTech Connect

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu . E-mail: jwdai@genetics.ac.cn

    2005-12-16

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.

  4. Antiadopogenic effects of rice hull smoke extract in 3T3-L1 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study investigates the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 pre-adipocyte cells. At concentrations of 0.1% and 0.5% RHSE, MDI-induced cells were shown to reduce their cellular lipid content by about 72% and 88%, respectively, compared to ...

  5. CLOCK promotes 3T3-L1 cell proliferation via Wnt signaling.

    PubMed

    Zhu, Zhu; Hua, Bingxuan; Xu, Lirong; Yuan, Gongsheng; Li, Ermin; Li, Xiaobo; Sun, Ning; Yan, Zuoqin; Lu, Chao; Qian, Ruizhe

    2016-07-01

    Circadian genes control most of the physiological functions including cell cycle. Cell proliferation is a critical factor in the differentiation of progenitor cells. However, the role of Clock gene in the regulation of cell cycle via wingless-type (Wnt) pathway and the relationship between Clock and adipogenesis are unclear. We found that the circadian locomotor output cycles kaput (Clock) regulated the proliferation and the adipogenesis of 3T3-L1 preadipocytes. We found that Clock attenuation inhibited the viability of 3T3-L1 preadipocytes in the cell counting kit 8. The expression of c-Myc and Cyclin D1 decreased dramatically in 3T3-L1 when Clock was silenced with short interfering RNA and was also decreased in fat tissue and adipose tissue-derived stem cells of Clock(Δ19) mice. Clock directly controls the expression of the components of Wnt signal transduction pathway, which was verified by serum shock, chromatin immunoprecipitation, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, IWR-1, a Wnt signal pathway inhibitor, inhibited the cell cycle promotion by CLOCK, which was detected by cell viability assay, flow cytometry, and qRT-PCR. Therefore, CLOCK transcription control of Wnt signaling promotes cell cycle progression in 3T3-L1 preadipocytes. Clock inhibited the adipogenesis on day 2 in 3T3-L1 cells via Oil Red O staining and qRT-PCR detection and probably related to cellular differentiation. These data provide evidence that the circadian gene Clock regulates the proliferation of preadipocytes and affects adipogenesis. © 2016 IUBMB Life, 68(7):557-568, 2016. PMID:27194636

  6. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  7. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  8. Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation

    PubMed Central

    Freytag, Svend O.; Utter, Merton F.

    1980-01-01

    The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res. Commun. 79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit Mr = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [14C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin. Images PMID:6929488

  9. RA induces the neural-like cells generated from epigenetic modified NIH/3T3 cells.

    PubMed

    Zhang, Xi-Mei; Li, Qiu-Ming; Su, Dong-Ju; Wang, Ning; Shan, Zhi-Yan; Jin, Lian-Hong; Lei, Lei

    2010-03-01

    Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells. PMID:19263240

  10. Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

    PubMed

    Alvares, Keith; Ren, Yinshi; Feng, Jian Q; Veis, Arthur

    2016-01-01

    P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and β-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study. PMID:26581835

  11. Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes

    PubMed Central

    Lee, Joomin; Kim, Ah-Ra; Lee, Jae-Joon

    2016-01-01

    The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts. PMID:26954122

  12. Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes.

    PubMed

    Lee, Joomin; Kim, Ah-Ra; Lee, Jae-Joon

    2016-09-01

    The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts. PMID:26954122

  13. Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells.

    PubMed Central

    Rozengurt, E; Sinnett-Smith, J

    1983-01-01

    Bombesin is shown to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations the peptide markedly enhances the ability of fresh serum to stimulate DNA synthesis in confluent and quiescent cultures of these cells. In the presence of a low concentration (3.5%) of serum, bombesin stimulates 3T3 cell proliferation. In serum-free medium, bombesin induces DNA synthesis in the absence of any other added growth factor; half-maximal effect is obtained at 1 nM. The mitogenic effect of bombesin is dependent on dose and time, is mimicked by litorin, and is markedly potentiated by insulin, colchicine, platelet-derived growth factor, and fibroblast-derived growth factor. These mitogens increase the maximal response elicited by bombesin and decrease the bombesin concentration required to produce half-maximal effect (from 1 nM to 0.3 nM). In contrast, vasopressin, phorbol esters, or cAMP increasing agents fail to enhance the maximal level of DNA synthesis induced by bombesin. Bombesin and litorin may provide useful model peptides for studies on the mechanism(s) by which extracellular ligands control cell proliferation. PMID:6344074

  14. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wu, Junyi

    Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

  15. Recommended protocol for the BALB/c 3T3 cell transformation assay.

    PubMed

    Sasaki, Kiyoshi; Bohnenberger, Susanne; Hayashi, Kumiko; Kunkelmann, Thorsten; Muramatsu, Dai; Phrakonkham, Pascal; Poth, Albrecht; Sakai, Ayako; Salovaara, Susan; Tanaka, Noriho; Thomas, B Claire; Umeda, Makoto

    2012-04-11

    The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA. PMID:22212201

  16. Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

    PubMed Central

    Mercier, N; Moldes, M; El Hadri, K; Fève, B

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

  17. Effect of Mangiferin and Mahanimbine on Glucose Utilization in 3T3-L1 cells

    PubMed Central

    Kumar, B Dinesh; Krishnakumar, K; Jaganathan, Saravana Kumar; Mandal, Mahitosh

    2013-01-01

    Background: Stem barks of Mangifera indica contain a rich content of mangiferin (xanthone glucoside), whereas Murraya koenigii leaves contain rich sources of mahanimbine (carbazole alkaloid) and used traditionally for the treatment of diabetes. Objective: To investigate the effects of mangiferin (xanthone glucoside) and mahanimbine (carbazole alkaloid) on glucose utilization in 3T3-L1 cells. Materials and Methods: Mangiferin was isolated from stem barks of Mangifera indica and mahanimbine was isolated from Murraya koenigii leaves. These isolated compounds were subjected to MTT assay and glucose utilization test with 3T3-L1 cells. Results: Treatment of the 3T3-L1 cells with mangiferin and mahanimbine increased the glucose utilization in a dose-dependent manner. At a concentration of 1 mM, mangniferin showed 2-fold increase in glucose utilization compared with untreated control. In case of mahanimbine, the observed effect at 1 mM was almost equivalent to positive control (insulin at 1 μM). Moreover, MTT assay showed that both of these compounds were less toxic at a concentration of 1 mM (nearly 75% cells are viable). Conclusion: The present results indicated that these natural products (mangiferin and mahanimbine) exhibited potential ethnomedical uses in management of diabetes. PMID:23661997

  18. EPAS1 promotes adipose differentiation in 3T3-L1 cells.

    PubMed

    Shimba, Shigeki; Wada, Taira; Hara, Shuntaro; Tezuka, Masakatsu

    2004-09-24

    Adipose differentiation is regulated by several transcription factors, such as the CAAT/enhancer-binding protein family and peroxisome proliferator activator (PPAR) gamma2. Several recent studies have shown that the basic helix-loop-helix-PAS superfamily is also involved in the regulation of adipose differentiation. In this study, we investigated the roles played by EPAS1 (endothelial PAS domain protein 1) in adipogenesis. EPAS1, also referred to as hypoxia-inducible factor 2alpha, is a transcription factor known to play essential roles in catecholamine homeostasis, vascular remodeling, and the maintenance of reactive oxygen species, and so forth. During adipose differentiation in 3T3-L1 cells, the level of EPAS1 mRNA began to increase 6 days after the induction, and EPAS1 was highly expressed in differentiated cells. To examine whether EPAS1 is involved in adipogenesis, we first isolated stable clones from 3T3-L1 cells in which we could induce the expression of an EPAS1 C-terminal deletion mutant (designated EPAS1-(1-485)) with the insect hormone. The induction of EPAS1-(1-485) allowed the cells to accumulate only minimum amounts of intracellular lipid droplets. Consistent with the morphological observations, a minimum amount of aP2 and PPARgamma2 mRNA was induced in the EPAS1-(1-485) cells. We then examined whether or not EPAS1 was able to promote adipogenesis in NIH 3T3 cells, a relatively nonadipogenic cell line. Overexpression of EPAS1 in NIH 3T3 cells induced a significant amount of lipid accumulation compared with that of the control cells in the presence of the PPARgamma ligand. The results were also confirmed by measuring the expression of adipocyte-related genes. Adenovirus-mediated EPAS1-(1-485) expression resulted in the reduction of basal and insulin-dependent glucose transport in 3T3-L1 adipocytes. The mechanism involved the transcriptional regulation of GLUT1, GLUT4, and IRS3 expression by EPAS1. Taken together, these results suggest that EPAS1 plays

  19. Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

    PubMed Central

    Yeh, W C; Bierer, B E; McKnight, S L

    1995-01-01

    Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479942

  20. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.

    PubMed

    Masiello, Lisa M; Fotos, Joseph S; Galileo, Deni S; Karin, Norman J

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1. PMID:16487757

  1. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  2. Vinculin expression in MC3T3-E1 cells in response to mechanical stimulus

    PubMed Central

    Cora-Cruz, J.J.; Diffoot-Carlo, N.; Sundaram, P.A.

    2015-01-01

    Loading frequency is known to influence the expression of the focal adhesions of the adherent cells. A small cyclical tensile force was transmitted to mouse pre-osteoblast MC3T3-E1 cells through PDMS substrates of varying stiffness. Changes in cell behavior with respect to proliferation and characteristics of focal adhesions were quantified through immunofluorescence labeling of vinculin. Amount of inactive vinculin was higher on substrates subjected to cyclic stimulation when compared with the results of the static substrates, whereas the number and area of focal adhesion points underwent a reduction. Inactive vinculin appears as a cloud in the cytoplasm in the vicinity of the nucleus. PMID:26858974

  3. Occurrence and control of sporadic proliferation in growth arrested Swiss 3T3 feeder cells.

    PubMed

    Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

    2015-01-01

    Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and

  4. The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

    PubMed Central

    Markovic, Jelena; Mora, Nancy J.; Broseta, Ana M.; Gimeno, Amparo; de-la-Concepción, Noelia; Viña, José; Pallardó, Federico V.

    2009-01-01

    Background Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate. Principal Findings We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation. Conclusions Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle. PMID:19641610

  5. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells - a murine acute myelomonocytic leukemia cell line - we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. PMID:25911323

  6. Poly(L-lactide) crystallization topography directs MC3T3-E1 cells response.

    PubMed

    Li, Wenqiang; Lu, Lu; Jiao, Yanpeng; Zhang, Chaowen; Zhou, Changren

    2016-09-01

    Biomaterial surface topography significantly influences cellular form and function. Using poly(L-lactic acid) films with normal spherulites, banded spherulites, and amorphous surfaces as model substrates, we conducted a systematic assessment of the role for polymer crystallization induced surface morphologies on cell growth and contact guidance. Microscopy and image analysis showed that the MC3T3-E1 cells spread out in a random fashion on the amorphous substrate. At 24 h post-seeding, MC3T3-E1 cells on both types of spherulite surfaces were elongated and aligned along the spherulite radius direction. For the banded spherulite surface with radial stripes and coupling annular grooves, the cell orientation and cell nuclear localization were related to the grooves structure. With increasing time, this orientation preference was weaker. These results demonstrate that the patterning of polymer crystallization structure provide important signals for guiding cells to exhibit characteristic orientation and morphology especially in the early stages of regeneration. PMID:27376548

  7. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Rozengurt, E.

    1985-11-01

    Gastrin-releasing peptide (GRP) labeled with /sup 125/I at tyrosine-15 (/sup 125/I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. /sup 125/I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells.

  8. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    SciTech Connect

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-02-19

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor {gamma}2, CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBP{beta} or C/EBP{delta}, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

  9. Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

    SciTech Connect

    Zhang Yuping; Tang Yongsong; Chen Xushen; Xu Peilin

    2007-09-10

    Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.

  10. Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells.

    PubMed

    Ishii, Ikumi; Ikeguchi, Yoshihiko; Mano, Hiroshi; Wada, Masahiro; Pegg, Anthony E; Shirahata, Akira

    2012-02-01

    Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis. PMID:21809076

  11. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  12. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H2O2 or TNFα was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  13. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. )

    1991-06-01

    The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

  14. Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

    PubMed

    Hou, Qingxia; Wang, Minglian; Wu, Shuicai; Ma, Xuemei; An, Guangzhou; Liu, Huan; Xie, Fei

    2015-03-01

    To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells. PMID:24665905

  15. Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

    PubMed

    Hirose, H; Suhara, T; Kaji, N; Sakuma, N; Sekijima, M; Nojima, T; Miyakoshi, J

    2008-01-01

    A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation. PMID:17694516

  16. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.

    PubMed Central

    Wu, J; Issa, J P; Herman, J; Bassett, D E; Nelkin, B D; Baylin, S B

    1993-01-01

    Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility. Images Fig. 1 Fig. 2 PMID:8415627

  17. Segmenting time-lapse phase contrast images of adjacent NIH 3T3 cells.

    PubMed

    Chalfoun, J; Kociolek, M; Dima, A; Halter, M; Cardone, A; Peskin, A; Bajcsy, P; Brady, M

    2013-01-01

    We present a new method for segmenting phase contrast images of NIH 3T3 fibroblast cells that is accurate even when cells are physically in contact with each other. The problem of segmentation, when cells are in contact, poses a challenge to the accurate automation of cell counting, tracking and lineage modelling in cell biology. The segmentation method presented in this paper consists of (1) background reconstruction to obtain noise-free foreground pixels and (2) incorporation of biological insight about dividing and nondividing cells into the segmentation process to achieve reliable separation of foreground pixels defined as pixels associated with individual cells. The segmentation results for a time-lapse image stack were compared against 238 manually segmented images (8219 cells) provided by experts, which we consider as reference data. We chose two metrics to measure the accuracy of segmentation: the 'Adjusted Rand Index' which compares similarities at a pixel level between masks resulting from manual and automated segmentation, and the 'Number of Cells per Field' (NCF) which compares the number of cells identified in the field by manual versus automated analysis. Our results show that the automated segmentation compared to manual segmentation has an average adjusted rand index of 0.96 (1 being a perfect match), with a standard deviation of 0.03, and an average difference of the two numbers of cells per field equal to 5.39% with a standard deviation of 4.6%. PMID:23126432

  18. The Role of Nuclear Factor-E2-Related Factor 1 in the Oxidative Stress Response in MC3T3-E1 Osteoblastic Cells

    PubMed Central

    Kim, Sung Hoon; Yoon, Hyun Koo; Yim, Chang Hoon

    2016-01-01

    Background Reactive oxygen species (ROS) and antioxidants are associated with maintenance of cellular function and metabolism. Nuclear factor-E2-related factor 1 (NFE2L1, Nrf1) is known to regulate the expression of a number of genes involved in oxidative stress and inflammation. The purpose of this study was to examine the effects of NFE2L1 on the response to oxidative stress in osteoblastic MC3T3-E1 cells. Methods The murine calvaria-derived MC3T3-E1 cell line was exposed to lipopolysaccharide (LPS) for oxidative stress induction. NFE2L1 effects were evaluated using small interfering RNA (siRNA) for NFE2L1 mRNA. ROS generation and the levels of known antioxidant enzyme genes were assayed. Results NFE2L1 expression was significantly increased 2.4-fold compared to the control group at 10 µg/mL LPS in MC3T3-E1 cells (P<0.05). LPS increased formation of intracellular ROS in MC3T3-E1 cells. NFE2L1 knockdown led to an additional increase of ROS (20%) in the group transfected with NFE2L1 siRNA compared with the control group under LPS stimulation (P<0.05). RNA interference of NFE2L1 suppressed the expression of antioxidant genes including metallothionein 2, glutamatecysteine ligase catalytic subunit, and glutathione peroxidase 1 in LPS-treated MC3T3-E1 cells. Conclusion Our results suggest that NFE2L1 may have a distinct role in the regulation of antioxidant enzymes under inflammation-induced oxidative stress in MC3T3-E1 osteoblastic cells. PMID:27118276

  19. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells

    PubMed Central

    Yoo, Yeong-Min; Han, Tae-Young; Kim, Han Sung

    2016-01-01

    Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis. PMID:27070587

  20. MC3T3-E1 Cell Response to Pure Titanium, Zirconia and Nano-Hydroxyapatite

    NASA Astrophysics Data System (ADS)

    Lee, Dong-Hwan; Han, Jung-Suk; Yang, Jae-Ho; Lee, Jai-Bong; Kim, Dae-Joon

    Titanium, zirconia and HAp were known as good biocompatible materials for tissue engineering. Osteblastic cell response is influence by the surface topography of material and its chemical composition as well. To evaluate the influence of different chemical compositions on osteoblast-like cells the specimens were polished until they have almost identical surface roughness. The commercially pure titanium, zirconia/alumina composite and nano-sized hydroxyapatite (HAp) specimens synthesized by hydrothermal method were used to evaluate the cell attachment, proliferation and differentiation. Confocal laser microscopy was used measurement of surface roughness, and flourescence microscopy and SEM were used to evaluate initial cell attachment and morphology after 3 hours. MTS assay was performed for cell proliferation after 1, 3, 7 days and ALP assay was used for cell differentiation after 7, 10, 14 days of cell culture period. Surface topography of nano-HAp specimen was almost identical compared with those of titanium and zirconia specimen. Under this condition, proliferation and differentiation of MC3T3-E1 cells was not significantly different with those on titanium and zirconia specimen. However, cells on Nano-HAp specimen showed quicker and more active cellular reaction for attachment when measured by the expression of adhesion proteins through confocal laser microscopy. The results suggested that the new nano-sized HAp can be applied as a suitable material for skeletal tissue engineering.

  1. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. )

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  2. Anthraquinones from Morinda officinalis roots enhance adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Liu, Qing; Kim, Seon Beom; Ahn, Jong Hoon; Hwang, Bang Yeon; Kim, Sung Yeon; Lee, Mi Kyeong

    2012-01-01

    To search for anti-diabetic and insulin-sensitising natural products, the effect on adipocyte differentiation was investigated by assessing fat accumulation in 3T3-L1 preadipocytes using Oil Red O staining. Fractionation and separation of n-hexane and CHCl₃ fractions of Morinda officinalis (Rubiaceae) using several chromatographic methods led to the isolation of three anthraquinones, 1,2-dimethoxyanthraquinone (1), alizarin-2-methyl ether (2) and rubiadin-1-methyl ether (3). Among them, alizarin-2-methyl ether (2) showed the strongest enhancing activity, followed by rubiadin-1-methyl ether (3) and 1,2-dimethoxyanthraquinone (1). At a concentration of 100 µM, alizarin-2-methyl ether (2) enhanced adipocyte differentiation by up to 131% (compared to insulin-treated cells). Thus, these compounds could be beneficial in the treatment of diabetes. PMID:22008000

  3. Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells.

    PubMed

    Inoue, Chisato; Kozaki, Tomomi; Morita, Yukiko; Shirouchi, Bungo; Fukami, Katsuya; Shimizu, Kuniyoshi; Sato, Masao; Katakura, Yoshinori

    2015-08-01

    Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood. PMID:25672941

  4. Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay.

    PubMed

    Sighinolfi, G L; Artoni, E; Gatti, A M; Corsi, L

    2016-05-01

    Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3 O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3 O4 ones, similar to those obtained by the positive control treatment (MCA + TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA + TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3 O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models. PMID:25358123

  5. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

    SciTech Connect

    Sheu, C.W.; Moreland, F.M.; Lee, J.K.; Dunkel, V.C.

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 /sup +/g/ml, did not induce transformation, whereas dioxane was very active in the induction type II foci in the cultured BALB/3T3 cells.

  6. Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation

    PubMed Central

    Wang, Yu; Cui, Haitao; Wu, Zhenxu; Wu, Naipeng; Wang, Zongliang; Chen, Xuesi; Wei, Yen; Zhang, Peibiao

    2016-01-01

    Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases. PMID:27149625

  7. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

    PubMed Central

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung

    2015-01-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

  8. Most short DNA molecules isolated from 3T3 cells are not nascent.

    PubMed Central

    Kowalski, J; Denhardt, D T

    1978-01-01

    The population of short DNA molecules (less than 10(3) nucleotides) in 3T3 cells has been studied using in vivo and in vitro pulse labeling techniques and in vitro end-labeling. There is a large number of molecules of less than 100 nucleotides present in equal numbers in both Go and S phase cells. In S phase cells, most of these molecules are not replicating intermediates because they do not become density-labeled after a moderate period of substitution of BrdUMP, although they are detected by end-labeling in vitro. This population includes the nascent Okazaki pieces that can be labeled in a short pulse with [3H]dThd or [3H]dTTP, however, these represent less than 10% of the total population. Alkaline hydrolysis of the molecules that had been end-labeled with 32P using [gamma32P]ATP and polynucleotide kinase did not reveal significant release of [32P] 2'(3'), 5' ribonucleoside diphosphates. PMID:724517

  9. Protein kinase C activation by anthracyclines in Swiss 3T3 cells.

    PubMed

    Lanzi, C; Gambetta, R A; Perego, P; Banfi, P; Franzi, A; Guazzoni, L; Zunino, F

    1991-01-01

    The effects of the anti-cancer anthracyclines doxorubicin and daunorubicin on the activity of protein kinase C (PKC) were examined in intact Swiss 3T3 cells. The 2 drugs stimulated the phosphorylation of an 80K phosphoprotein found to be identical to that generated in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate as indicated by gel electrophoresis and peptide mapping. The effect of doxorubicin was dose-dependent in the range 10(-5) to 10(-3) M and was not associated with a detectable translocation of PKC activity from cytosol to the cell membrane. Doxorubicin and daunorubicin were found to increase the incorporation of phosphate into phosphatidic acid, phosphatidylinositol 4-monophosphate and phosphatidyl inositol 4,5-bisphosphate. In addition, the anthracyclines induced a rise in inositol phosphates, thus indicating a stimulation of the breakdown of phosphoinositides. These data are consistent with an indirect mechanism of PKC activation by anthracyclines. We propose that diacylglycerol, which is derived from the hydrolysis of phospholipids, (including the phosphoinositides), by activation of phospholipases, could mediate PKC activation. The described effects, involving cell-signal-transducing pathways, emphasize a new aspect of the cellular actions of these anti-tumor agents. PMID:1845961

  10. Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies▿ †

    PubMed Central

    Cheng, Chiang-Min; Li, Huiling; Gasman, Stéphane; Huang, Jian; Schiff, Rachel; Chang, Eric C.

    2011-01-01

    Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively. PMID:21189290

  11. A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor.

    PubMed

    Moore, J G; Fuchs, C A; Hata, Y S; Hicklin, D J; Colucci, G; Chrispeels, M J; Feldman, M

    2000-07-26

    A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation. PMID:10913819

  12. Induction of Adipocyte Differentiation by Polybrominated Diphenyl Ethers (PBDEs) in 3T3-L1 Cells

    PubMed Central

    Tung, Emily W. Y.; Boudreau, Adèle; Wade, Michael G.; Atlas, Ella

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBPα, PPARγ, and LXRα were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis. PMID:24722056

  13. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    SciTech Connect

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-05-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio ((activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)). Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of /sup 125/I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

  14. Hindered Diffusion of Inert Tracer Particles in the Cytoplasm of Mouse 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Luby-Phelps, Katherine; Castle, Philip E.; Lansing Taylor, D.; Lanni, Frederick

    1987-07-01

    Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 angstrom in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 angstrom, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.

  15. (-)-Ternatin inhibits adipogenesis and lipid metabolism in 3T3-L1 cells.

    PubMed

    Ito, Masahiko; Ito, Junko; Kitazawa, Hidefumi; Shimamura, Ken; Fukami, Takehiro; Tokita, Shigeru; Shimokawa, Kenichiro; Yamada, Kaoru; Kanatani, Akio; Uemura, Daisuke

    2009-06-01

    (-)-Ternatin, a highly N-methylated cyclic peptide, inhibits fat accumulation in 3T3-L1 cells and reduces fat mass in mice. However, the mechanism for its anti-adipogenic effect has remained unknown. To examine the mechanism used by (-)-ternatin to inhibit adipocyte differentiation, we examined the effects of (-)-ternatin and [l-Ala(4)]ternatin, an inactive analog of (-)-ternatin, on the expression of adipocyte markers and lipogenic enzymes. We found that (-)-ternatin potently reduced mRNA expression of several adipocyte markers in a dose-dependent manner, whereas [l-Ala(4)]ternatin showed no effects. At the immediate early phase, (-)-ternatin, but not [l-Ala(4)]ternatin, reduced the expression of Srebp1c, Fas, Acc2 and C/EBP-alpha while showing no effects on C/EBP-beta and C/EBP-delta. These results suggest that (-)-ternatin affects the mid-to late differentiation stages of adipocytes. Consistent with the decreased expression of lipogenic enzymes, (-)-ternatin potently inhibited triglyceride synthesis. Intriguingly, (-)-ternatin also inhibited triglyceride synthesis in rat primary hepatocytes, suggesting that the potential action sites for (-)-ternatin are shared by adipocytes and liver. Although the target molecule of (-)-ternatin remains unknown, our data suggest that (-)-ternatin and its potential target might provide a new therapeutic approach to metabolic disorders. PMID:19463739

  16. Cellular uptake and fate of fibroin microspheres loaded with randomly fragmented DNA in 3T3 cells

    PubMed Central

    Lee, Jin Sil; Hur, Won

    2016-01-01

    Purified fibroin protein can be obtained in large quantities from silk fibers and processed to form microscopic particles as delivery vehicles for therapeutic agents. In this study, we demonstrated that fibroin microspheres were taken up by 3T3 cells, localized in the nonlysosomal compartment, and secreted from the cytoplasm after medium replenishment. DNA-loaded microspheres were taken up by >95% of 3T3 cells. DNA cargo had no influence on the intracellular trafficking of microspheres, while fluorescently labeled cargo DNA was observed in the lysosomal compartment and in the microspheres. These results indicate that fibroin microspheres can travel through 3T3 cells without making any contact with the lysosomal compartments. The amount of DNA loaded in the microspheres taken up by 3T3 cells was estimated up to 831.0 pg/cell. Thus, fibroin microspheres can deliver a large amount of randomly fragmented DNA (<10 kb) into the cytoplasmic compartment of 3T3 cells. PMID:27257379

  17. STAT5A expression in Swiss 3T3 cells promotes adipogenesis in vivo in an athymic mice model system.

    PubMed

    Stewart, William C; Pearcy, Lisa A; Floyd, Z Elizabeth; Stephens, Jacqueline M

    2011-09-01

    Many studies from our laboratories and others have shown that STAT5 expression and activity are increased during adipogenesis of murine and human adipocytes. Ectopic expression of STAT5A in fibroblasts or preadipocytes can confer or enhance adipogenesis. To determine whether STAT5A also plays a role in adipogenesis in vivo, we injected athymic mice with Swiss 3T3 cells expressing an empty pBABE retrovirus, Swiss cells expressing a pBABE retrovirus-containing STAT5A, or 3T3-F442A preadipocytes. Athymic mice injected with either 3T3-F442A cells or Swiss 3T3 cells expressing STAT5A resulted in fat pad formation at the site of injection. However, mice injected with Swiss cells containing the parent retroviral vector did not have any observable fat pads. An analysis of the ectopic fat pads obtained from the Swiss 3T3 STAT5A mice revealed abundant expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin. The protein levels of both of these fat cell markers were comparable to expression levels in epididymal fat pads. These results demonstrate that STAT5A can promote adipogenesis in vivo in this model system which supports a role of this transcription factor in adipocyte development in the whole animal. PMID:21494231

  18. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  19. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  20. Exogenous Sodium Pyruvate Stimulates Adipogenesis of 3T3-L1 Cells.

    PubMed

    Hwang, Ji-Sun; Kim, Song-Yi; Jung, Eun-Hye; Kwon, Mi-Youn; Kim, Kyoung-Hong; Cho, Hyeongjin; Han, Inn-Oc

    2016-01-01

    We investigated the effects of exogenous sodium pyruvate (SP) on adipocyte differentiation, lipid accumulation, and the mRNA expression levels of adipogenesis-related genes in 3T3-L1 pre-adipocytes. Differentiation of pre-adipocytes was induced by MDI (3-isobutyl-1-methylxanthine: IBMX, dexamethasone: DEX, and insulin), in the presence or absence of SP. Adipogenesis was stimulated by SP in a concentration-dependent manner. SP also induced the expression of genes encoding aP2, GLUT4, and adiponectin, but had no effect on cell proliferation. Exogenous glucose did not promote adipogenesis or lipid accumulation. 2-deoxy-D-glucose inhibited adipogenesis initiated by MDI, but failed to influence the effects of SP on adipogenesis, whereas 3-bromopyruvate inhibited adipogenesis regardless of whether SP was present. The pro-adipogenic properties of SP were limited to the early events of adipogenesis. To determine whether SP mimics the adipogenic action of dexamethasone or insulin, we examined the effects of SP on adipogenesis with combinations of IBMX, DEX, and insulin. SP did not improve incomplete lipid accumulation observed in cells grown under IBMX-, DEX-, or insulin-free conditions. Insulin-stimulated ERK1/2 phosphorylation was diminished by SP, while phosphorylation of Akt was increased, correlating with increased glucose uptake in response to insulin. We also observed that SP stimulated immediate early expression of C/EBPβ and C/EBPδ. The PPARγ antagonist GW9662 inhibited adipogenesis. Our findings highlight the adipogenic function of exogenous SP by stimulating early events of adipogenesis. PMID:26053972

  1. Shp2 suppresses the adipogenic differentiation of preadipocyte 3T3-L1 cells at an early stage

    PubMed Central

    Tao, J; Zheng, L; Meng, M; Li, Y; Lu, Z

    2016-01-01

    Tyrosine phosphatase protein Shp2 is a potential therapeutic target for obesity. However, the mechanism of Shp2 during adipogenesis is not fully understood. The present study investigated the role of Shp2 in the terminal differentiation of preadipocytes. The results showed that Shp2 suppressed adipocyte differentiation in 3T3-L1 cells; overexpression of Shp2 reduced lipid droplet production in 3T3-L1 cells, whereas Shp2 knockdown increased lipid droplet production in 3T3-L1 cells. Furthermore, inhibition of Shp2 activity also enhanced adipocyte differentiation. Interestingly, Shp2 expression was specifically decreased early during differentiation in response to stimulation with the dexamethasone–methylisobutylxanthine–insulin (DMI) hormone cocktail. During the first 2 days of differentiation, Shp2 overexpression impaired the DMI-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in 3T3-L1 cells and blocked the peak expression of CCAAT/enhancer-binding proteins β and δ during preadipocyte differentiation. In conclusion, Shp2 downregulated the early stages of hormone-induced differentiation of 3T3-L1 cells and inhibited the expression of the first wave of transcription factors by suppressing the DMI-induced STAT3 signaling pathway. These discoveries point to a novel role of Shp2 during adipogenesis and support the hypothesis that Shp2 could be a therapeutic target for the control of obesity. PMID:27551539

  2. Lithium differentially affects clock gene expression in serum-shocked NIH-3T3 cells.

    PubMed

    Osland, Teresa M; Fernø, Johan; Håvik, Bjarte; Heuch, Ivar; Ruoff, Peter; Lærum, Ole Didrik; Steen, Vidar M

    2011-07-01

    Bipolar disorder has been associated with disturbances in circadian rhythms. Lithium is frequently used in the long-term treatment of bipolar disorder, and has been shown to prolong such rhythms in animals and humans. To examine whether lithium affects the expression of genes regulating the circadian clock, cultured NIH-3T3 cells were synchronized by serum-shocking, and the relative expression of the clock genes Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), Cryptochrome2 (Cry2), Brain and muscle aryl hydrocarbon nuclear translocator-like 1 (Bmal1), Circadian locomotor output cycles kaput (Clock), Rev-Erb-α (Nr1d1), RAR-related orphan receptor α (Ror-α), Glycogen synthase kinase-3β (Gsk-3β), Casein kinase 1-ε (CK1-ε; Csnk1ε), E4 binding protein 4 (E4BP4; Nfil-3) and albumin D-binding protein (Dbp) was examined for three consecutive days in the presence of lithium (20 mM) or vehicle (20 mM NaCl). We found that lithium significantly increased the expression of Per2 and Cry1, whereas Per3, Cry2, Bmal1, E4BP4 and Rev-Erb-α expression was reduced. We also found that lithium prolonged the period of Per2. Taken together, these effects on clock gene expression may be relevant for the effects of lithium on biological rhythms and could also give new leads to further explore its mood-stabilizing actions in the treatment of bipolar disorder. PMID:20837565

  3. High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis.

    PubMed

    Wang, Xiaoju; Feng, Zhengping; Li, Jiling; Chen, Lixue; Tang, Weixue

    2016-07-01

    In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis

  4. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells

    PubMed Central

    Henein, Alexandra E.; Hanlon, Geoffrey W.; Cooper, Callum J.; Denyer, Stephen P.; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 109 pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required. PMID:27536286

  5. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells.

    PubMed

    Henein, Alexandra E; Hanlon, Geoffrey W; Cooper, Callum J; Denyer, Stephen P; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 10(9) pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required. PMID:27536286

  6. Prolonged treatment with 3-isobutyl-1-methylxanthine improves the efficiency of differentiating 3T3-L1 cells into adipocytes.

    PubMed

    Hua, Yongjie; Ke, Shanshan; Wang, Yao; Irwin, David M; Zhang, Shuyi; Wang, Zhe

    2016-08-15

    Until now, the low efficiency of current protocols or kits for the differentiation of 3T3-L1 preadipocytes makes it difficult to continue the studies of the cellular and molecular mechanisms in adipocytes. Here we present a productive and highly efficient protocol for the differentiation of 3T3-L1 cells that uses a prolonged treatment with 3-isobutyl-1-methylxanthine (IBMX) during the differentiated process. 3T3-L1 cells of unknown passage +3 and unknown passage +7 treated with a prolonged exposure to IBMX showed significantly increased differentiation efficiency by day 15, in contrast to low levels of differentiation seen with protocols that lacked additional IBMX. PMID:27210514

  7. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    SciTech Connect

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-02-15

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

  8. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing Liao, Er-Yuan

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  9. Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells

    SciTech Connect

    Sagara, Chiaki; Takahashi, Katsuhiko; Kagechika, Hiroyuki; Takahashi, Noriko

    2013-03-29

    Highlights: ► We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ► 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ► A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ► This antagonist had no effects on RXRα and PPARγ levels in 9-cis-RA-treated cells. ► 9-cis-RA-induced decrease in both RXRα and PPARγ was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor α (RXRα) with peroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXRα and PPARγ. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPARγs levels in a RXR activation-independent manner.

  10. 6-gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid droplets in 3T3-L1 cells.

    PubMed

    Tzeng, Thing-Fong; Liu, I-Min

    2013-04-15

    6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). In this study, we investigated the effects of 6-gingerol on the inhibition of adipogenesis in 3T3-L1 cells. After treatment with 6-gingerol in differentiation medium for 4 or 8 days, the 3T3-L1 cells were lysed for experimental analysis. Cells were stained with Oil-Red-O to detect oil droplets in adipocytes. The 3T3-L1 cells were lysed and measured for triglyceride contents. The protein expression of adipogenesis-related transcription factor was evaluated by Western blot analysis. 6-Gingerol suppressed oil droplet accumulation and reduced the droplet size in a concentration (5-15 μg/ml)- and time-dependent manner. Treatment of 3T3-L1 cells with 6-gingerol reduced the protein levels of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)α. Additionally, the protein levels of fatty acid synthase (FAS) and adipocyte-specific fatty acid binding protein (aP2) decreased upon treatment with 6-gingerol. Meanwhile, 6-gingerol diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9). These results suggest that 6-gingerol effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of PPARγ and C/EBPα and subsequently inhibits FAS and aP2 expression. 6-Gingerol also inhibited differentiation in 3T3-L1 cells by attenuating the Akt/GSK3β pathway. Our findings provide important insights into the mechanisms underlying the anti-adipogenic activity of 6-gingerol. PMID:23369342

  11. Osmotically inducible uptake of betaine via amino acid transport system A in SV-3T3 cells.

    PubMed

    Petronini, P G; De Angelis, E; Borghetti, A F; Wheeler, K P

    1994-05-15

    The osmotically inducible uptake of betaine (NNN-trimethylglycine) by SV-3T3 cells has been studied and compared with the similar process in MDCK cells. Betaine uptake by SV-3T3 cells could be described in terms of a saturable, Na(+)-dependent, component plus a small non-saturable, Na(+)-independent, component. Transport was active, producing considerable accumulation of betaine in the cells. After exposure of the cells to hypertonic conditions for 6 h, there was a marked increase in betaine uptake. Kinetic analysis indicated that this increase resulted from an increase in the Vmax. value of the saturable component, from about 88 to 185 nmol of betaine/5 min per mg of protein, the corresponding Km values of about 15 and 10 mM not being significantly different. This induction of transport activity was detectable only after about 2 h exposure of the cells to hypertonic medium, closely paralleling an induction of influx of N-methylaminoisobutyric acid, and was prevented by the presence of cycloheximide. Betaine influx was markedly inhibited by several neutral amino acids, particularly those transported by system A, such as N-methylaminoisobutyric acid and the imino acid proline. A high concentration (25 mM) of betaine also significantly inhibited the uptake of proline by SV-3T3 cells. Although very similar results were obtained with MDCK cells, prolonged exposure of cells to hypertonic conditions revealed distinct differences. When the hypertonic incubation was extended from 6 h to 24 h, betaine transport in SV-3T3 cells either remained the same or decreased, whereas it showed a further marked increase in MDCK cells, and also became sensitive to inhibition by gamma-aminobutyric acid. mRNA for the betaine transporter BGT-1 [Yamauchi, Uchida, Kwon, Preston, Brooks Robey, Garcia-Perez, Burg and Handler (1992) J. Biol. Chem. 267, 649-652] was detectable in MDCK cells exposed to hypertonic medium for 24 h, but not in SV-3T3 cells under any conditions. It is concluded that

  12. A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

    NASA Astrophysics Data System (ADS)

    Pan, Hsu-An; Hung, Yao-Ching; Su, Chia-Wei; Tai, Shih-Ming; Chen, Chiun-Hsun; Ko, Fu-Hsiang; Steve Huang, G.

    2009-08-01

    Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.

  13. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    PubMed Central

    Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

  14. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats

    NASA Astrophysics Data System (ADS)

    Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

    2012-04-01

    While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 μg mL-1, 10 μg mL-1, 100 μg mL-1, 1 mg mL-1, and 10 mg mL-1 were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL-1) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg-1 body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair.

  15. Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.

    PubMed Central

    Moutsatsos, I K; Wade, M; Schindler, M; Wang, J L

    1987-01-01

    Proliferating 3T3 mouse fibroblasts contain higher levels of the lectin carbohydrate-binding protein 35 (CBP35) than do quiescent cultures of the same cells. An immunofluorescence study was carried out with a rabbit antiserum directed against CBP35 to map the cellular fluorescence distribution in a large population of cells under different growth conditions. This cytometric analysis showed that the lectin is predominantly localized in the nucleus of the proliferating cells. In quiescent 3T3 cultures, the majority of the cells lost their nuclear staining and underwent a general decrease in the overall fluorescence intensity. Stimulation of serum-starved quiescent 3T3 cells by the addition of serum resulted in an increase in the level of CBP35. The percentage of cells showing distinct punctate intranuclear staining reached a maximum at about the same time as the onset of the first S-phase of the cell cycle. All of these results suggest that CBP35 may be a protein whose presence in the nucleus, in discrete punctate distribution, is coordinated with the proliferation state of the cell. Images PMID:3306680

  16. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko; Nishio, Hiroaki

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  17. FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Eda, Homare; Aoki, Katsuhiko; Marumo, Keishi; Fujii, Katsuyuki; Ohkawa, Kiyoshi

    2008-02-08

    Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPAR{gamma}. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

  18. Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold.

    PubMed

    Koç, Aysel; Elçin, Ayşe Eser; Elçin, Yaşar Murat

    2016-09-01

    This study evaluates the suitability of a macroporous three-dimensional chitosan/hydroxyapatite (CS/HA) composite as a bone tissue engineering scaffold using MC3T3-E1 cells. The CS/HA scaffold was produced by freeze-drying, and characterized by means of SEM and FTIR. In vitro findings demonstrated that CS/HA supported attachment and proliferation of cells, and stimulated extracellular matrix (ECM) production. Tissue biocompatibility and osteogenic capacity of the cell-laden constructs were evaluated in an ectopic Wistar rat model. In vivo results showed that the MC3T3-E1 cell-laden CS/HA was essentially histocompatible, promoted neovascularization and calcified matrix formation, and secreted osteoblast-specific protein. We conclude that the composite scaffold evaluated has potential for applications in bone regeneration. PMID:25968048

  19. Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

    PubMed

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

  20. Nickel-Refining Fumes Induced DNA Damage and Apoptosis of NIH/3T3 Cells via Oxidative Stress

    PubMed Central

    Wang, Yue; Wang, Sheng-Yuan; Jia, Li; Zhang, Lin; Ba, Jing-Chong; Han, Dan; Yu, Cui-Ping; Wu, Yong-Hui

    2016-01-01

    Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. PMID:27347984

  1. Bombesin stimulation of c-fos and c-myc gene expression in cultured of Swiss 3T3 cells

    SciTech Connect

    Palumbo, A.P.; Rossino, P.; Comoglio, P.M.

    1986-11-01

    Bombesin has been show to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations it stimulates DNA synthesis in quiescent cultures of 3T3 cells and also induces the expression of c-fos and c-myc mRNA. c-fos mRNA transcripts dramatically increase 15 min after the addition of bombesin, are still abundant after 30-60 min and then decrease. c-myc mRNA induction is detectable later, 1 h after bombesin treatment. Conversely, no changes in c-Ki-ras expression are observed after stimulation with bombesin. These results demonstrate that the increased expression of c-fos and c-myc mRNAs appears to be a common response to diverse agents that induce DNA synthesis and cell proliferation.

  2. Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity.

    PubMed

    Hammer, Bruce E; Kidder, Louis S; Williams, Philip C; Xu, Wayne Wenzhong

    2009-11-01

    Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed. PMID:20052306

  3. Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells

    SciTech Connect

    Kim, Soon-hee; Park, Hee-Sook; Lee, Myoung-su; Cho, Yong-Jin; Kim, Young-Sup; Hwang, Jin-Taek; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young

    2008-07-18

    Inhibition of adipocyte differentiation is one approach among the anti-obesity strategies. This study demonstrates that vitisin A, a resveratrol tetramer, inhibits adipocyte differentiation most effectively of 18 stilbenes tested. Fat accumulation and PPAR{gamma} expression were decreased by vitisin A in a dose-dependent manner. Vitisin A significantly inhibited preadipocyte proliferation and consequent differentiation within the first 2 days of treatment, indicating that the anti-adipogenic effect of vitisin A was derived from anti-proliferation. Based on cell cycle analysis, vitisin A blocked the cell cycle at the G1-S phase transition, causing cells to remain in the preadipocyte state. Vitisin A increased p21 expression, while the Rb phosphorylation level was reduced. Therefore, vitisin A seems to induce G1 arrest through p21- and consequent Rb-dependent suppression of transcription. On the other hand, ERK and Akt signaling pathways were not involved in the anti-mitotic regulation by vitisin A. Taken together, these results suggest that vitisin A inhibits adipocyte differentiation through preadipocyte cell cycle arrest.

  4. Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in 3T3-L1 Preadipocyte Cell Line.

    PubMed

    Tutino, Valeria; Orlando, Antonella; Russo, Francesco; Notarnicola, Maria

    2016-02-01

    The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, α-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. PMID:26189725

  5. The effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation.

    PubMed

    Gagnon, A; Sorisky, A

    1998-03-01

    We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0.1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1.7-fold greater, and insulin-stimulated phosphoinositide 3-kinase association with IRS-1 was 2.3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses. PMID:9545023

  6. Long-term exposure of 3T3 fibroblast cells to endocrine disruptors alters sensitivity to oxidative injury.

    PubMed

    Nishimura, Yuka; Nakai, Yasuyoshi; Tanaka, Aiko; Nagao, Tetsuji; Fukushima, Nobuyuki

    2014-07-01

    When Swiss 3T3 fibroblasts were exposed to bisphenol A (BPA) or nonylphenol (NP) within a range of 0.1-100 nM for 30-45 days, increased resistance to oxidative injury was found. Western blot analysis indicated concomitant increased expression of bcl-2 protein and reduced histone methylation levels in cells after BPA or NP exposure. Using a heterologous expression system, both chemicals could stimulate G protein-coupled receptor 30 (GPR30), a transmembrane estrogen receptor predominantly expressed in 3T3 cells, at lower concentrations, which gave increased survival. Taken together, these results suggest that BPA or NP exposure might cause alterations in cellular activity against oxidative stress, possibly through GPR30. PMID:24604882

  7. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

  8. Puerarin enhances adipocyte differentiation, adiponectin expression, and antioxidant response in 3T3-L1 cells.

    PubMed

    Lee, Ok-Hwan; Seo, Dong-Ho; Park, Cheon-Seok; Kim, Young-Cheul

    2010-01-01

    Puerarin, a major isoflavone glycoside from Kudzu root (Pueraria lobata), has been reported to exert antihyperglycemic and antioxidant effects and thus have pharmacological actions in the treatment of diabetes and cardiovascular diseases. We investigated the effects of puerarin on the changes of key gene expression associated with adipocyte differentiation and insulin sensitivity and link to cellular antioxidant response pathways. Puerarin treatment significantly enhanced differentiation of 3T3-L1 preadipocytes accompanying increased lipid accumulation and glucose-6-phosphate dehydrogenase (G6PDH) activity. At a molecular level, puerarin upregulated mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes, an adipocyte-specific fatty acid binding protein (aP2) and GLUT4. Puerarin also caused a significant increase in mRNA level of adiponectin, an important insulin-sensitizing adipocytokine that is downregulated in insulin-resistant and diabetic states. In addition, treatment with puerarin was found to upregulate mRNA levels of G6PDH, glutathione reductase, and catalase, all of which are important for endogenous antioxidant responses. These data suggest that the hypoglycemic effects of puerarin can be attributed to the upregulation of PPARγ and its downstream target genes, GLUT4 and adiponectin expression, leading to increased glucose utilization. Puerarin may also be effective in preventing the rise of oxidative stress during adipocyte differentiation by increasing endogenous antioxidant responses. PMID:20806284

  9. Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

    NASA Technical Reports Server (NTRS)

    Lin, H. S.; Hughes-Fulford, M.; Kumegawa, M.; Pitts, A. C.; Snowdowne, K. W.

    1993-01-01

    We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

  10. Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay.

    PubMed

    Sasaki, Kiyoshi; Bohnenberger, Susanne; Hayashi, Kumiko; Kunkelmann, Thorsten; Muramatsu, Dai; Poth, Albrecht; Sakai, Ayako; Salovaara, Susan; Tanaka, Noriho; Thomas, B Claire; Umeda, Makoto

    2012-04-11

    This catalogue is a display of focus photos representative of the BALB/c 3T3 cell transformation assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results. PMID:22331008

  11. Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype.

    PubMed Central

    Wei, L Y; Stutts, M J; Hoffman, M M; Roepe, P D

    1995-01-01

    Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR. Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs. Two other clones expressing lower levels of CFTR are less resistant. As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline. Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different. Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression. CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake. These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR. Images FIGURE 1 FIGURE 3 FIGURE 6 PMID:8519988

  12. Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

    2002-01-01

    Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

  13. 4-Hydroxyderricin, as a PPARγ Agonist, Promotes Adipogenesis, Adiponectin Secretion, and Glucose Uptake in 3T3-L1 Cells.

    PubMed

    Li, Yongjia; Goto, Tsuyoshi; Yamakuni, Kanae; Takahashi, Haruya; Takahashi, Nobuyuki; Jheng, Huei-Fen; Nomura, Wataru; Taniguchi, Masahiko; Baba, Kimiye; Murakami, Shigeru; Kawada, Teruo

    2016-07-01

    Adipocyte differentiation plays a pivotal role in maintaining the production of small-size adipocytes with insulin sensitivity, and impaired adipogenesis is implicated in insulin resistance. 4-Hydroxyderricin (4-HD), a phytochemical component of Angelica keiskei, possesses diverse biological properties such as anti-inflammatory, antidiabetic, and antitumor. In the present study, we investigated the effects of 4-HD on adipocyte differentiation. 4-HD promoted lipid accumulation in 3T3-L1 cells, upregulated both peroxisome proliferator-activated receptor (PPAR)-γ mRNA and protein expression, and acted as a ligand for PPARγ in the luciferase assay. Moreover, 4-HD increased the mRNA and protein expression levels of adiponectin. Additionally, it promoted insulin-dependent glucose uptake into 3T3-L1 adipocytes and increased Akt phosphorylation and glucose transporter (GLUT) 4 mRNA expression. In summary, these findings suggest that 4-HD, which promoted adipogenesis and insulin sensitivity in 3T3-L1 cells, might be a phytochemical with potent insulin-sensitizing effects. PMID:27098252

  14. Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells.

    PubMed

    Xu, Qian; Liu, Wei; Liu, Xiaoling; Liu, Weiwei; Wang, Hongju; Yao, Guodong; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

    2016-09-01

    Primary cilium is a cellular antenna, signalling as a sensory organelle. Numerous pathological manifestation is associated with change of its length. Although the interaction between autophagy and primary cilia has been suggested, the role of autophagy in primary cilia length is largely unknown. In this study the primary cilia were immunostained and observed by using confocal fluorescence microscopy, and we found that silibinin, a natural flavonoid, shortened the length of primary cilia, meanwhile it also induced autophagy in 3T3-L1 cells. This study was designed to investigate the significance of silibinin-induced autophagy in primary ciliary structure in confluent mouse embryo fibroblast 3T3-L1 cells. Either blocking the autophagic flux with pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), or transfection of siRNA targeting LC3 inhibited the reduction of cilia length caused by silibinin exposure. Autophagy induced by silibinin decreased expressions of the cilia-associated proteins, such as IFT88, KIF3a and Ac-tubulin, while 3-MA restored it, indicating that autophagy induced by silibinin led to a reduction of primary cilia length. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy, was up-regulated by silibinin in a time-dependent manner. In addition, 3T3-L1 cells treated with siRNA against HDAC6 had a reduced autophagic level and were protected from silibinin-induced cilia shortening. Taken together, we conclude that the HDAC6-mediated autophagy negatively regulates primary cilia length during silibinin treatment and has the potential to serve as a therapeutic target for primary cilia-associated ciliopathies. These findings thus provide new information about the potential link between autophagy and primary cilia. PMID:27435857

  15. Isoflavones in Chickpeas Inhibit Adipocyte Differentiation and Prevent Insulin Resistance in 3T3-L1 Cells.

    PubMed

    Gao, Yue; Yao, Yang; Zhu, Yinging; Ren, Guixing

    2015-11-11

    Diabetes mellitus is a metabolic disease characterized by hyperglycemia arising from defects in insulin secretion. This study investigated the effects of isoflavones in chickpea sprouts germinated in light (IGL) and isoflavones in chickpea seeds (ICS) on insulin resistance through their role in suppression of 3T3-L1 adipocyte differentiation. Results showed that IGL and ICS inhibit the differentiation of 3T3-L1 pre-adipocytes induced by differentiation medium in a dose-dependent manner, and the suppressive effect of IGL was stronger (p < 0.05) than that of ICS, evidenced by a decrease of Oil Red O staining and intracellular triacylglycerol content in the mature adipocytes. IGL and ICS also stimulated glucose uptake significantly (p < 0.05). Besides, IGL and ICS treatment caused a significant decrease in mRNA and protein expression levels of adipogenesis-related transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα). Furthermore, the mRNA and protein expression levels of adipocyte fatty acid-binding protein (ap2), lipoprotein lipase (LPL), uncoupling protein-2 (UCP-2), and glucose transporter 4 (Glut4) in 3T3-L1 cells were also markedly down-regulated (p < 0.05). PMID:26494490

  16. Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells.

    PubMed

    Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A; Chock, P Boon

    2013-05-01

    Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT. PMID:23415771

  17. Restoration of murine femoral segmental defect using CTGF-overexpressing MC3T3-E1 cells

    PubMed Central

    Huang, Xiangyu; Li, Yanqiu; Xu, Jiantao; Liu, Kai; Yu, Xin; Cheng, Xin; Xu, Dongdong; Li, Zubing

    2016-01-01

    Connective tissue growth factor (CTGF) is a member of the CCN super family and is reported to widely participate in bone development and regeneration. This study aimed to restore murine femoral segmental defect using CTGF-overexpressing MC3T3-E1 cells. MC3T3-E1 cells were transinfected by lenti-CTGF (LvCTGF) and lenti-negative control (LvNC) virus to obtain stably transinfected cells. Real-time PCR, Western blot, alkaline phosphatase activity assay, and alizarin red staining demonstrated that the overexpression of CTGF enhanced osteogenesis in vitro. Cell migration assay results showed that LvCTGF cells expressed higher migration ability than LvNC cells, while CCK-8 assay revealed no significant difference in cell proliferation. The LvCTGF and LvNC cells were then seeded into a chitosan/β-TCP scaffold and were used to restore a murine femoral segmental defect. Samples were harvested by the end of 2 and 5 weeks respectively. Micro-CT analysis and Masson’s trichrome staining results showed that the LvCTGF-scaffold group expressed better bone healing compared with the LvNC-scaffold and scaffold-only groups. CTGF-overexpressed cells serve as an efficient source of seeding cells for bone regeneration. PMID:27186279

  18. Zinc deprivation impairs growth factor-stimulated calcium influx into murine 3T3 cells associated with decreased cell proliferation.

    PubMed

    O'Dell, Boyd L; Browning, Jimmy D

    2011-06-01

    Zinc plays a critical role in growth, a process that depends primarily on cell proliferation. Murine fibroblasts, Swiss 3T3 cells, were used to explore the hypothesis that a critical role of zinc in cell proliferation relates to its function in calcium influx. Cells were deprived of zinc by an impermeant chelator, diethylenetriaminepentaacetate (0.6 mmol/L), and low-calcium status was achieved by using a low- (<5 μmol/L) calcium medium. Cells were stimulated by a composite of growth factors (GF): platelet-derived GF, insulin-like GF-I, and epidermal GF. GF stimulation of cell proliferation was assessed by the incorporation of tritiated thymidine and calcium influx by the increase in fluorescence of cells loaded with Fluo-4. Proliferation was dependent on both zinc and calcium and they interacted in this process. GF stimulated an immediate sharp increase in intracellular calcium, indicative of internal calcium release, which peaked within 1 min and decreased to an elevated plateau, a pattern typical of a store-operated calcium channel. The sustained calcium influx of zinc-deprived cells was markedly lower than that of supplemented cells. Verapamil, a calcium channel blocker, also depressed both cell proliferation and calcium influx. In summary, zinc deficiency impaired GF-stimulated calcium influx into murine fibroblasts in association with decreased cell proliferation. PMID:21508206

  19. TNF-α Induces Caspase-1 Activation Independently of Simultaneously Induced NLRP3 in 3T3-L1 Cells.

    PubMed

    Furuoka, Mana; Ozaki, Kei-Ichi; Sadatomi, Daichi; Mamiya, Sayaka; Yonezawa, Tomo; Tanimura, Susumu; Takeda, Kohsuke

    2016-12-01

    The intracellular cysteine protease caspase-1 is critically involved in obesity-induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase-1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD-like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase-1 itself. However, it is not fully understood how caspase-1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF-α-induced activation of caspase-1 is accompanied by robust induction of NLRP3 in 3T3-L1 adipocytes but that caspase-1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3-L1 cells with TNF-α induced mRNA expression and activation of caspase-1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF-α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase-1 induced by TNF-α, suggesting that NLRP3 is dispensable for TNF-α-induced caspase-1 activation. Moreover, we did not detect the basal and TNF-α-induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase-1 activation. These results suggest that TNF-α induces caspase-1 activation in an inflammasome-independent manner in 3T3-L1 cells and that the ERK-dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761-2767, 2016. © 2016 Wiley Periodicals, Inc. PMID:26989816

  20. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    SciTech Connect

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. )

    1991-03-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

  1. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts. PMID:25904074

  2. Bioconversion of Citrus unshiu peel extracts with cytolase suppresses adipogenic activity in 3T3-L1 cells

    PubMed Central

    Lim, Heejin; Yeo, Eunju; Song, Eunju; Chang, Yun-Hee; Han, Bok-Kyung; Choi, Hyuk-Joon

    2015-01-01

    BACKGROUND/OBJECTIVES Citrus flavonoids have a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated whether bioconversion of Citrus unshiu with cytolase (CU-C) ameliorates the anti-adipogenic effects by modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 cells. MATERIALS/METHODS Glycoside forms of Citrus unshiu (CU) were converted into aglycoside forms with cytolase treatment. Cell viability of CU and CU-C was measured at various concentrations in 3T3L-1 cells. The anti-adipogenic and lipolytic effects were examined using Oil red O staining and free glycerol assay, respectively. We performed real time-polymerase chain reaction and western immunoblotting assay to detect mRNA and protein expression of adipogenic transcription factors, respectively. RESULTS Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and instead, increased flavanone aglycoside forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with CU or CU-C at a dose of 0.5 mg/ml. Adipocyte differentiation was inhibited in CU-C group, but not in CU group. CU-C markedly suppressed the insulin-induced protein expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) as well as the mRNA levels of CEBPα, PPARγ, and sterol regulatory element binding protein 1c (SREBP1c). Both CU and CU-C groups significantly increased the adipolytic activity with the higher release of free glycerol than those of control group in differentiated 3T3-L1 adipocytes. CU-C is particularly superior in suppression of adipogenesis, whereas CU-C has similar effect to CU on stimulation of lipolysis. CONCLUSIONS These results suggest that bioconversion of Citrus unshiu peel extracts with cytolase enhances aglycoside flavonoids and improves the anti-adipogenic metabolism via both inhibition of key adipogenic

  3. Berberine inhibits SREBP-1-related clozapine and risperidone induced adipogenesis in 3T3-L1 cells.

    PubMed

    Hu, Yueshan; Kutscher, Eric; Davies, Gareth E

    2010-12-01

    Weight gain is a common and potentially serious complication associated with the treatment of second generation antipsychotics such as clozapine and risperidone. Increased peripheral adipogenesis via the SREBP-1 pathway could be one critical mechanism responsible for antipsychotic drug-induced weight gain. Berberine, a botanical alkaloid, has been shown in our previous studies to inhibit adipogenesis in cell and animal models. MTT was used to determine the cytotoxic effects of clozapine and risperidone in combination with berberine. Differentiation of 3T3-L1 cells was monitored by Oil-Red-O staining and the expression of SREBP-1 and related proteins was determined by real-time RT-PCR and western blotting. The results showed that neither clozapine nor risperidone, alone or in combination with berberine had significant effects on cell viability. Eight days treatment with 15 μM clozapine increased adipogenesis by 37.4% and 50 μM risperidone increased adipogenesis by 26.5% during 3T3-L1 cell differentiation accompanied by increased SREBP-1, PPARγ, C/EBPα, LDLR and Adiponectin gene expression. More importantly, the addition of 8 μM berberine diminished the induction of adipogenesis almost completely accompanied by down-regulated mRNA and protein expression levels of SREBP-1-related proteins. These encouraging results may lead to the use of berberine as an adjuvant to prevent weight gain during second generation antipsychotic medication. PMID:20564506

  4. Characterization of RNA from Noninfectious Virions Produced by Sarcoma Positive-Leukemia Negative Transformed 3T3 Cells

    PubMed Central

    Phillips, Leo A.; Hollis, Vincent W.; Bassin, Robert H.; Fischinger, Peter J.

    1973-01-01

    RNA from noninfectious virions produced by two established clonal lines of sarcoma positive-leukemia negative (S+L-)-transformed 3T3 cells has been characterized. RNA from virions or nucleoids of S+L--(C243) cells consisted of three to four sizes: ±44 S (6%), 28 S (17%), 18 S (38%), and <18 S (39%). 28S virion RNA contained some virus-specific information demonstrable by RNA·DNA hybridization with a DNA probe derived from the RNA-directed DNA polymerase product of murine sarcoma-leukemia virus, while parallel studies indicated that 28S ribosomal RNA from ribosomal subunits of transformed and nontransformed 3T3 cells did not contain virus-specific information. In contrast to the S+L-(C243) virions, RNA from virions or nucleoids of S+L-(D56) cells consisted of five sizes: 80 S (6%), 68 S (8%), 56 S (5%), 28 S (28%), and <28 S (53%). Thermal dissociation studies suggested that this S+L- genome is comprised of 28S RNA subunits. From these studies we postulate that the 28S viral RNA is most probably the monomeric genome of S+L- virions. PMID:4355380

  5. Effects of Scytosiphon lomentaria on osteoblastic proliferation and differentiation of MC3T3-E1 cells

    PubMed Central

    Park, Mi Hwa; Kim, Seoyeon; Cheon, Jihyeon; Lee, Juyeong; Kim, Bo Kyung; Lee, Sang-Hyeon; Kong, Changsuk; Kim, Yuck Yong

    2016-01-01

    BACKGROUND/OBJECTIVES Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. PMID:27087897

  6. EGF raises cytosolic Ca sup 2+ in A431 and Swiss 3T3 cells by a dual mechanism

    SciTech Connect

    Pandiella, A.; Malgaroli, A.; Meldolesi, J.; Vicentini, L.M. )

    1987-05-01

    The changes in Ca{sup 2+} homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca{sup 2+} concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca{sup 2+}-containing medium caused a rapid rise in the cytosolic {sup 45}Ca{sup 2+} concentration, that remained elevated for several minutes. In Ca{sup 2+}-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca{sup 2+} signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the {sup 45}Ca{sup 2+} influx effect of EGF is under the control of a separate, as yet unidentified mechanism.

  7. Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells

    SciTech Connect

    Vara, F.; Schneider, J.A.; Rozengurt, E.

    1985-04-01

    Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive YWRb uptake, a measure of the activity of the Na /K pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt2) also enhanced ouabain sensitive YWRb uptake and amiloride-sensitive SSNa influx. Prolonged treatment (40 hr) of 3T3 cells with PBt2 at a saturating dose, which reduces the number of PBt2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt2. They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na /H antiport activity, which, in turn, leads to Na influx, intracellular pH modulation, and stimulation of the Na /K pump.

  8. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

    PubMed Central

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  9. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers.

    PubMed

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  10. A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

    PubMed

    Kono, Ryohei; Okuno, Yoshiharu; Inada, Ken-ichi; Tokuda, Akihiko; Hashizume, Hiroshi; Yoshida, Munehito; Nakamura, Misa; Utsunomiya, Hirotoshi

    2011-01-01

    Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis. PMID:21979066

  11. Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

    PubMed

    Babb, Rebecca C; Homer, Karen A; Robbins, Jon; Lax, Alistair J

    2012-01-01

    Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q). PMID:23144805

  12. Layer-by-Layer assembled growth factor reservoirs for steering the response of 3T3-cells.

    PubMed

    Naves, Alliny F; Motay, Marvin; Mérindol, Rémi; Davi, Christiane P; Felix, Olivier; Catalani, Luiz H; Decher, Gero

    2016-03-01

    Layer-by-Layer (LbL) assemblies of heparin (Hep) and chitosan (Chi) were prepared for use as reservoirs for acidic and basic fibroblast growth factors (aFGFs and bFGFs, respectively). The effects of the architecture and composition of the reservoirs on the viability and proliferation of NIH-3T3 fibroblast cells were studied under starvation conditions. The reservoir stability was monitored by ellipsometry. The aFGF and bFGF loadings were determined using a dissipation-enhanced quartz crystal microbalance (QCM-D). Stability and release assays were performed in a phosphate buffer at physiological conditions. The results demonstrated that the amount of aFGF and bFGF loaded into and released from LbL reservoirs composed of 3 and 6 layer pairs could be controlled. Cell culture assays in low serum culture medium (LSCM) demonstrated that incorporating very small amounts of aFGF and bFGF into the (Hep/Chi)n multilayers significantly improved the proliferation of the NIH-3T3 fibroblasts. The cells did not proliferate on (Hep/Chi)n assemblies prepared in the absence of FGF under identical conditions. The LbL reservoirs were highly effective for the long-term storage (up to 9 months) of aFGF and bFGF. This work demonstrates the potential of LbL reservoirs for use as biomaterial coatings. PMID:26700236

  13. 5,6-Dehydrokawain from Alpinia zerumbet promotes osteoblastic MC3T3-E1 cell differentiation.

    PubMed

    Kumagai, Momochika; Mishima, Takashi; Watanabe, Akio; Harada, Teppei; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Tawata, Shinkichi; Nishikawa, Keisuke; Morimoto, Yoshiki

    2016-07-01

    Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent. PMID:26940726

  14. The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells.

    PubMed

    Yang, Fang; Nakajima, Yoshihiro; Kumagai, Megumi; Ohmiya, Yoshihiro; Ikeda, Masaaki

    2009-02-27

    To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells. PMID:19138663

  15. Municipal wastewater affects adipose deposition in male mice and increases 3T3-L1 cell differentiation.

    PubMed

    Biasiotto, Giorgio; Zanella, Isabella; Masserdotti, Alice; Pedrazzani, Roberta; Papa, Matteo; Caimi, Luigi; Di Lorenzo, Diego

    2016-04-15

    Trace concentration of EDs (endocrine disrupting compounds) in water bodies caused by wastewater treatment plant effluents is a recognized problem for the health of aquatic organisms and their potential to affect human health. In this paper we show that continuous exposure of male mice from early development to the adult life (140 days) to unrestricted drinking of wastewater collected from a municipal sewage treatment plant, is associated with an increased adipose deposition and weight gain during adulthood because of altered body homeostasis. In parallel, bisphenol A (BPA) at the administration dose of 5 μg/kg/body weight, shows an increasing effect on total body weight and fat mass. In vitro, a solid phase extract (SPE) of the wastewater (eTW), caused stimulation of 3T3-L1 adipocyte differentiation at dilutions of 0.4 and 1 % in the final culture medium which contained a concentration of BPA of 40 nM and 90 nM respectively. Pure BPA also promoted adipocytes differentiation at the concentration of 50 and 80 μM. BPA effect in 3T3-L1 cells was associated to the specific activation of the estrogen receptor alpha (ERα) in undifferentiated cells and the estrogen receptor beta (ERβ) in differentiated cells. BPA also activated the Peroxisome Proliferator Activated Receptor gamma (PPARγ) upregulating a minimal 3XPPARE luciferase reporter and the PPARγ-target promoter of the aP2 gene in adipose cells, while it was not effective in preadipocytes. The pure estrogen receptor agonist diethylstilbestrol (DES) played an opposite action to that of BPA inhibiting PPARγ activity in adipocytes, preventing cell differentiation, activating ERα in preadipocytes and inhibiting ERα and ERβ regulation in adipocytes. The results of this work show that the drinking of chemically-contaminated wastewater promotes fat deposition in male mice and that EDs present in sewage are likely responsible for this effect through a nuclear receptor-mediated mechanism. PMID:26944108

  16. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    SciTech Connect

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-12-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

  17. Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

    PubMed Central

    Barnes, D W; Colowick, S P

    1977-01-01

    Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent. PMID:202958

  18. Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

    PubMed

    Barnes, D W; Colowick, S P

    1977-12-01

    Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent. PMID:202958

  19. DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells

    PubMed Central

    Thaler, Roman; Spitzer, Silvia; Karlic, Heidrun; Klaushofer, Klaus; Varga, Franz

    2012-01-01

    Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.  PMID:22507896

  20. Ivy gourd (Coccinia grandis L. Voigt) root suppresses adipocyte differentiation in 3T3-L1 cells

    PubMed Central

    2014-01-01

    Background Ivy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model. Methods Dried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels. Results Of the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPARγ, C/EBPα, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells. Conclusions This is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPARγ-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes. PMID:24884680

  1. Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals.

    PubMed

    Tanaka, Noriho; Bohnenberger, Susanne; Kunkelmann, Thorsten; Munaro, Barbara; Ponti, Jessica; Poth, Albrecht; Sabbioni, Enrico; Sakai, Ayako; Salovaara, Susan; Sasaki, Kiyoshi; Thomas, B Claire; Umeda, Makoto

    2012-04-11

    The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data

  2. The Effect of Bovine Parathyroid Hormone Withdrawal on MC3T3-E1 Cell Proliferation and Phosphorus Metabolism

    PubMed Central

    Li, Sijia; Cui, Tongxia; Li, Zhonghe; Zhang, Bin; Li, Zhuo; Wu, Jianxiong; Liang, Xinling; Lin, Zheng; Shi, Wei

    2015-01-01

    Hypocalcemia and hypophosphatemia are common complications after parathyroidectomy (PTX). Sudden removal of high circulating levels of parathyroid hormone (PTH) causes decreased osteoclastic resorption resulting in a decreased bone remodeling space. These phenomena are likely due to an increased influx of calcium and phosphorus into bone. However, there are currently no data to support this hypothesis. In this study, we found that PTX significantly reduced levels of PTH, calcium and phosphate. Compared with preoperative levels, after 1 year, postoperative PTH, calcium and phosphate levels were 295.6 ± 173.7 pg/mL (P < 0.05), 86.62 ± 15.98 mg/dL (P < 0.05) and 5.56 ± 2.03 mg/dL (P < 0.05), respectively. We investigated continuous bovine PTH administration as well as withdrawal of bovine PTH stimulation in the mouse osteoblast precursor cell line MC3T3-E1. MC3T3-E1 cells were cultured with continuous bovine PTH treatment for 20 days or with transient bovine PTH treatment for 10 days. High doses of continuous bovine PTH exposure strongly reduced cell proliferation, alkaline phosphatase activity and the number of mineralized calcium nodules. However, withdrawal of bovine PTH (100 ng/mL) significantly increased the number of mineralized calcium nodules and caused a rapid decline in calcium and phosphorus content of culture medium. In conclusion, continuous exposure to bovine PTH inhibited osteoblast differentiation and reduced the formation of mineralized nodules. However, this inhibition was removed and mineralized nodule formation resumed with withdrawal of bovine PTH. According to the results of our clinical examinations and in vitro experiments, we hypothesize that the sudden removal of high levels of PTH may cause an increased influx of calcium and phosphorus into bone after PTX. PMID:25775025

  3. The effect of bovine parathyroid hormone withdrawal on MC3T3-E1 cell proliferation and phosphorus metabolism.

    PubMed

    Liu, Shuangxin; Zhu, Weiping; Li, Sijia; Cui, Tongxia; Li, Zhonghe; Zhang, Bin; Li, Zhuo; Wu, Jianxiong; Liang, Xinling; Lin, Zheng; Shi, Wei

    2015-01-01

    Hypocalcemia and hypophosphatemia are common complications after parathyroidectomy (PTX). Sudden removal of high circulating levels of parathyroid hormone (PTH) causes decreased osteoclastic resorption resulting in a decreased bone remodeling space. These phenomena are likely due to an increased influx of calcium and phosphorus into bone. However, there are currently no data to support this hypothesis. In this study, we found that PTX significantly reduced levels of PTH, calcium and phosphate. Compared with preoperative levels, after 1 year, postoperative PTH, calcium and phosphate levels were 295.6 ± 173.7 pg/mL (P < 0.05), 86.62 ± 15.98 mg/dL (P < 0.05) and 5.56 ± 2.03 mg/dL (P < 0.05), respectively. We investigated continuous bovine PTH administration as well as withdrawal of bovine PTH stimulation in the mouse osteoblast precursor cell line MC3T3-E1. MC3T3-E1 cells were cultured with continuous bovine PTH treatment for 20 days or with transient bovine PTH treatment for 10 days. High doses of continuous bovine PTH exposure strongly reduced cell proliferation, alkaline phosphatase activity and the number of mineralized calcium nodules. However, withdrawal of bovine PTH (100 ng/mL) significantly increased the number of mineralized calcium nodules and caused a rapid decline in calcium and phosphorus content of culture medium. In conclusion, continuous exposure to bovine PTH inhibited osteoblast differentiation and reduced the formation of mineralized nodules. However, this inhibition was removed and mineralized nodule formation resumed with withdrawal of bovine PTH. According to the results of our clinical examinations and in vitro experiments, we hypothesize that the sudden removal of high levels of PTH may cause an increased influx of calcium and phosphorus into bone after PTX. PMID:25775025

  4. [Effects of isoquercitrin from Craibiodendron yunnanense on osteogenic differentiation of MC3T3-E1 cells].

    PubMed

    Duan, Ai-Zhu; Deng, Xu-Liang; Li, Rong-Tao

    2014-10-01

    Natural products especially flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. The present study is to investigate the effects of isoquercitrin from Craibiodendron yunnanense with different concentrations at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) on proliferation, differentiation and mineralization of MC3T3-E1. Cell proliferation was assessed by CCK-8 kit at 1, 3, 5 and 7 days of culture. Alkaline phosphatase (ALP) activity were performed qualitatively and quantitatively on day 7, and alizarin red S staining was employed to access the mineralization of cells on day 21. The osteogenic markers ALP, collagen type I (COL 1A1), runt-related transcription factor 2 (Runx-2) and Osterix were detected to analysis early osteogenic differentiation of cells on day 3 by RT-PCR. The results showed that isoquercitrin had a dose-dependent effect on the proliferation, osteogenic differentiation, mineralization and gene expression of MC3T3-E1 in the range from 1 x 10(-7) to 1 x 10(-5) mol x L(-1). At concentrations above 1 x 10(-4) mol x L(-1) isoquercitrin showed cytotoxicity, while 1 x 10(-6) mol x L(-1) is the optimal concentration of isoquercitrin to improve the osteoblastic activity. All these results implied that isoquercitrin might be the major composition of traditional Chinese medicine C. yunnanense to treat bone fractures. PMID:25612450

  5. Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells.

    PubMed

    Rasmussen, Line Jee Hartmann; Müller, Helene Steenkær Holm; Jørgensen, Bente; Pedersen, Stine Falsig; Hoffmann, Else Kay

    2015-01-15

    The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process. PMID:25377086

  6. Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction.

    PubMed

    Kunoh, Tatsuki; Wang, Weixiang; Kobayashi, Hiroaki; Matsuzaki, Daisuke; Togo, Yuki; Tokuyama, Masahiro; Hosoi, Miho; Koseki, Koichi; Wada, Shu-Ichi; Nagai, Nobuo; Nakamura, Toshinobu; Nomura, Shintaro; Hasegawa, Makoto; Sasaki, Ryuzo; Mizukami, Tamio

    2015-01-01

    Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell-cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy. PMID:26284361

  7. Characterization of the respiration of 3T3 cells by laser-induced fluorescence during a cyclic heating process

    NASA Astrophysics Data System (ADS)

    Beuthan, J.; Dressler, C.; Zabarylo, U.; Minet, O.

    2010-04-01

    The use of lasers in the near infrared spectral range for laser-induced tumor therapy (LITT) demands a new understanding of the thermal responses to repetitive heat stress. The analysis of laser-induced fluorescence during vital monitoring offers an excellent opportunity to solve many of the related issues in this field. The laser-induced fluorescence of the cellular coenzyme NADH was investigated for its time and intensity behavior under heat stress conditions. Heat was applied to vital 3T3 cells (from 22°C to 50°C) according to a typical therapeutical time regime. A sharp increase in temperature resulted in non-linear time behavior when the concentration of this vital coenzyme changed. There are indications that biological systems have a delayed reaction on a cellular level. These results are therefore important for further dosimetric investigations.

  8. Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction

    PubMed Central

    Kunoh, Tatsuki; Wang, Weixiang; Kobayashi, Hiroaki; Matsuzaki, Daisuke; Togo, Yuki; Tokuyama, Masahiro; Hosoi, Miho; Koseki, Koichi; Wada, Shu-ichi; Nagai, Nobuo; Nakamura, Toshinobu; Nomura, Shintaro; Hasegawa, Makoto; Sasaki, Ryuzo; Mizukami, Tamio

    2015-01-01

    Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy. PMID:26284361

  9. Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation.

    PubMed

    Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

    2016-09-15

    Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0-15μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. PMID:27475717

  10. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    PubMed

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-01-01

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. PMID:25279585

  11. MC3T3-E1 cell response to stainless steel 316L with different surface treatments.

    PubMed

    Zhang, Hongyu; Han, Jianmin; Sun, Yulong; Huang, Yongling; Zhou, Ming

    2015-11-01

    In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4h, 1d, 3d, 7d), and cell proliferation was assessed by MTT method at 1d, 3d, and 7d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1h. The polished sample was smooth (Sq: 1.8nm), and the blasted and HA coated samples were much rougher (Sq: 3.2μm and 7.8μm). Within 1d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3d and 7d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1d and 3d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7d. Protein adsorption on the HA coated samples was the highest at 1h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. PMID:26249561

  12. T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

    PubMed

    Ray, Durwood B; Merrill, Gerald A; Brenner, Frederic J; Lytle, Laurie S; Lam, Tan; McElhinney, Aaron; Anders, Joel; Rock, Tara Tauber; Lyker, Jennifer Kier; Barcus, Scott; Leslie, Kara Hust; Kramer, Jill M; Rubenstein, Eric M; Pryor Schanz, Karen; Parkhurst, Amy J; Peck, Michelle; Good, Kimberly; Granath, Kristi Lemke; Cifra, Nicole; Detweiler, Jessalee Wantz; Stevens, Laura; Albertson, Richard; Deir, Rachael; Stewart, Elisabeth; Wingard, Katherine; Richardson, Micah Rose; Blizard, Sarah B; Gillespie, Lauren E; Kriley, Charles E; Rzewnicki, Daniel I; Jones, David H

    2016-01-01

    Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7

  13. Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies.

    PubMed

    Poburski, Doerte; Thierbach, René

    2016-01-01

    The identification of cancer preventive or therapeutic substances as well as carcinogenic risk assessment of chemicals is nowadays mostly dependent on animal studies. In vitro cell transformation assays mimic different stages of the in vivo neoplastic process and represent an excellent alternative to study carcinogenesis and therapeutic options. In the BALB/c-3T3 two-stage transformation assay cells are chemically transformed by treatment with MCA and TPA, along with the final Giemsa staining of morphological aberrant foci. In addition to the standard method we can show, that it is possible to apply other chemicals in parallel to identify potential preventive or therapeutic substances during the transformation process. Furthermore, we successfully combined the BALB/c cell transformation assay with several endpoint applications for protein analysis (immunoblot, subcellular fractionation and immunofluorescence) or energy parameter measurements (glucose and oxygen consumption) to elucidate cancer mechanisms in more detail. In our opinion the BALB/c cell transformation assay proves to be an excellent model to investigate alterations in key proteins or energy parameters during the different stages of transformation as well as therapeutic substances and their mode of action. PMID:27611302

  14. Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

    PubMed Central

    Poburski, Doerte; Thierbach, René

    2016-01-01

    The identification of cancer preventive or therapeutic substances as well as carcinogenic risk assessment of chemicals is nowadays mostly dependent on animal studies. In vitro cell transformation assays mimic different stages of the in vivo neoplastic process and represent an excellent alternative to study carcinogenesis and therapeutic options. In the BALB/c-3T3 two-stage transformation assay cells are chemically transformed by treatment with MCA and TPA, along with the final Giemsa staining of morphological aberrant foci. In addition to the standard method we can show, that it is possible to apply other chemicals in parallel to identify potential preventive or therapeutic substances during the transformation process. Furthermore, we successfully combined the BALB/c cell transformation assay with several endpoint applications for protein analysis (immunoblot, subcellular fractionation and immunofluorescence) or energy parameter measurements (glucose and oxygen consumption) to elucidate cancer mechanisms in more detail. In our opinion the BALB/c cell transformation assay proves to be an excellent model to investigate alterations in key proteins or energy parameters during the different stages of transformation as well as therapeutic substances and their mode of action. PMID:27611302

  15. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

    PubMed Central

    Koenig, B B; Cook, J S; Wolsing, D H; Ting, J; Tiesman, J P; Correa, P E; Olson, C A; Pecquet, A L; Ventura, F; Grant, R A

    1994-01-01

    The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images PMID:8065329

  16. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    PubMed

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

    2016-09-01

    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. PMID:27427305

  17. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  18. MC3T3-E1 Cells Behavior on Surfaces Bombarded by Argon Ions in Planar Cathode Discharge.

    PubMed

    Moura, Carlos Eduardo Bezerra; Silva, Naisandra Bezerra; Sa, Juliana Carvalho; Cavalcanti, Geraldo Barroso; de Medeiros, Silvia Regina Batistuzzo; Rocha, Hugo Alexandre Oliveira; Papa, Paula Carvalho; Alves, Clodomiro

    2016-05-01

    To evaluate the effect of topography in nanoscale, titanium surfaces were bombarded by argon ions (a chemically inert gas), in an atmosphere of plasma. The effects of surface parameters on morphology, adhesion, proliferation, and MC3T3-E1 preosteoblasts differentiation were analyzed. Nontreated (smooth) surfaces were used as a control. The levels of average roughness (Ra) observed in bombarded and smooth titanium surfaces were of 95 and 14 nm, respectively. The wettability increased on treated surfaces. The number of attached cells (30 and 60 min) was significantly higher on the bombarded surface. The cell proliferation after 3 and 7 days was also significantly higher on the ion-bombarded surface. In addition, the ALP activity and expression of osteocalcin were higher in cells grown on the treated surface. The results showed that bombardment with argon ions increased the roughness and the wettability of the Ti surface, promoting a significant increase in the adhesion, proliferation, and differentiation of preosteoblasts. PMID:26416762

  19. Deoxyactein stimulates osteoblast function and inhibits bone-resorbing mediators in MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2013-03-01

    Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic agents with minimal side effects is highly desirable. Cimicifuga racemosa has a long and diverse history of medicinal use and deoxyactein isolated from this species is one of the major constituents. In the present study, the effect of deoxyactein on the function of osteoblastic MC3T3-E1 cells was studied. Deoxyactein caused a significant elevation of cell growth, alkaline phosphatase activity, collagen content, and mineralization in the cells (P < 0.05). Moreover, deoxyactein significantly (P < 0.05) decreased the production of reactive oxygen species (ROS) and osteoclast differentiation-inducing factors such as TNF-α, IL-6 and receptor activator of nuclear factor-κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. These results demonstrate that deoxyactein may have positive effects on skeletal structure. PMID:21910134

  20. S6 kinase in quiescent Swiss mouse 3T3 cells is activated by phosphorylation in response to serum treatment

    SciTech Connect

    Ballou, L.M.; Siegmann, M.; Thomas, G. )

    1988-10-01

    To investigate the role of phosphorylation in the activation of S6 kinase, the enzyme was isolated from {sup 32}P-labeled Swiss mouse 3T3 cells before and after stimulation with serum. The kinase activity was followed through several purification steps, and a radioactive protein of M{sub r} 70,000 was obtained from the stimulated cells. This band was not detected in resting cells. The M{sub r} 70,000 protein exhibited the same size upon NaDodSO{sub 4}/PAGE as the homogeneous kinase, and it comigrated with the in vitro autophosphorylated form of the enzyme. Treatment of the in vivo-labeled material with phosphatase 2A led to a loss of kinase activity concomitant with a release of {sup 32}P{sub i} from the M{sub r} 70,000 protein. The partially dephosphorylated protein migrated faster during PAGE, displaying distinct species of M{sub r} 69,000 and 68,000. Most importantly, phospho amino acid analysis of the labeled S6 kinase showed only phosphoserine and phosphothreonine. These results argue that the S6 kinase is phosphorylated at multiple sites in vivo and that it is activated by serine/threonine phosphorylation.

  1. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    PubMed

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  2. Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

    PubMed

    Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

    2013-09-01

    In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. PMID:23820182

  3. Protective effect of quercitrin against hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2012-03-01

    The protective effect of quercitrin on the response of osteoblastic MC3T3-E1 cells to oxidative stress was evaluated. Osteoblasts were incubated with H(2)O(2) and/or quercitrin, and markers of osteoblast function and oxidative damage were examined. Quercitrin treatment reversed the cytotoxic effect of H(2)O(2) significantly (P<0.05). This effect was blocked by ICI182780 and LY294002, suggesting that quercitrin's effect might be involved in estrogen action and results from PI3K mediated signaling pathway. Pretreatment of quercitrin increased collagen content, alkaline phosphatase (ALP) activity, and calcium deposition of osteoblasts compared with H(2)O(2) treated cells and these effects were blocked by ERKs and p38 mitogen-activated protein kinases (MAPKs) inhibitors such as PD98059 and SB203580, respectively. These suggest that quercitrin-induced protective effect against osteoblast dysfunction by oxidative stress is associated with increased activation of ERKs and p38 MAPK. Pretreatment with quercitrin also reduced the increase in bone-resorbing factor, receptor activator of nuclear factor-kB ligand (RANKL) and oxidative damage markers (malondialdehyde, protein carbonyl, and nitrotyrosine) induced by H(2)O(2). These results suggest that quercitrin may be protective against H(2)O(2)-induced dysfunction in osteoblasts. PMID:20822887

  4. Adipogenesis, lipogenesis and lipolysis is stimulated by mild but not severe hypoxia in 3T3-L1 cells.

    PubMed

    Weiszenstein, Martin; Musutova, Martina; Plihalova, Andrea; Westlake, Katerina; Elkalaf, Moustafa; Koc, Michal; Prochazka, Antonin; Pala, Jan; Gulati, Sumeet; Trnka, Jan; Polak, Jan

    2016-09-16

    In-vitro investigation of the effects of hypoxia is limited by physical laws of gas diffusion and cellular O2 consumption, making prolonged exposures to stable O2 concentrations impossible. Using a gas-permeable cultureware, chronic effects of mild and severe hypoxia on triglyceride accumulation, lipid droplet size distribution, spontaneous lipolysis and gene expression of adipocyte-specific markers were assessed. 3T3-L1 cells were differentiated under 20%, 4% or 1% O2 using a gas-permeable cultureware. Triglyceride accumulation, expression of genes characteristic for advanced adipocyte differentiation and involvement of key lipogenesis enzymes were assessed after exposures. Lipogenesis increased by 375% under mild hypoxia, but dropped by 43% in severe hypoxia. Mild, but not severe, hypoxia increased formation of large lipid droplets 6.4 fold and strongly induced gene expression of adipocyte-specific markers. Spontaneous lipolysis increased by 488% in mild, but only by 135% in severe hypoxia. Inhibition of ATP-dependent citrate lyase suppressed hypoxia-induced lipogenesis by 81% and 85%. Activation of HIF inhibited lipogenesis by 59%. Mild, but not severe, hypoxia stimulates lipolysis and promotes adipocyte differentiation, probably through excess of acetyl-CoA originating from tricarboxylic acid cycle independently of HIF activation. PMID:27498031

  5. The Effect of OSM on MC3T3-E1 Osteoblastic Cells in Simulated Microgravity with Radiation

    PubMed Central

    Goyden, Jake; Tawara, Ken; Hedeen, Danielle; Willey, Jeffrey S.; Thom Oxford, Julia; Jorcyk, Cheryl L.

    2015-01-01

    Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor α subunits, collagen α1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss. PMID:26030441

  6. Cytotoxicity of zinc oxide nanoparticles on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells.

    PubMed

    Pandurangan, Muthuraman; Veerappan, Muthuviveganandavel; Kim, Doo Hwan

    2015-02-01

    The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3-L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells. PMID:25380643

  7. Oroxylin A, a constituent of Oroxylum indicum inhibits adipogenesis and induces apoptosis in 3T3-L1 cells.

    PubMed

    Singh, Jyotsna; Kakkar, Poonam

    2014-10-15

    Oroxylin A (OA) is a flavonoid found in Oroxylum indicum, a medicinal plant with multiple biological activities. This study was taken up to investigate the effect of OA, on adipogenesis, lipolysis and apoptosis in 3T3 L1 cells. Pre-adipocytes were treated with 10-40 μM OA on various days of adipogenesis treatment schedule. Mature adipocytes were treated with OA for lipolysis and apoptosis studies. In maturing pre-adipocytes, 10 μM OA suppressed intracellular lipid accumulation by 42.19% which was confirmed by lipidTox imaging of cells. In addition, OA decreased the nuclear translocation of PPARγ and mRNA expression of its downstream genes (FAS and LPL) along with adiponectin secretion. In mature adipocytes, 40 μM of OA decreased cell viability by 30% of control. Annexin V/PI staining showed induction of apoptosis which was further confirmed by enhanced levels of pro-apoptotic proteins Bax, cyt c, AIF and chromatin condensation. OA enhanced TNF-α secretion, lipolysis and decreased Akt phosphorylation in mature adipocytes. Findings suggest that OA possibly exerts its anti-obesity effect by affecting adipocyte life cycle at critical points of differentiation and maturity. When we compared the potency of OA with non-methoxylated flavonoids morin, naringenin and kaempferol on adipocyte life cycle OA was far more potent. Thus, study clearly indicates a new role for oroxylin A as regulator of adipocyte life cycle. In addition, study also suggested a specific role of methoxylated group in exerting lipolysis and cytotoxic effects in mature adipocytes. PMID:25442284

  8. Fucoxanthin exerts differing effects on 3T3-L1 cells according to differentiation stage and inhibits glucose uptake in mature adipocytes

    SciTech Connect

    Kang, Seong-Il; Ko, Hee-Chul; Shin, Hye-Sun; Kim, Hyo-Min; Hong, Youn-Suk; Lee, Nam-Ho; Kim, Se-Jae

    2011-06-17

    Highlights: {yields} Fucoxanthin enhances 3T3-L1 adipocyte differentiation at an early stage. {yields} Fucoxanthin inhibits 3T3-L1 adipocyte differentiation at intermediate and late stages. {yields} Fucoxanthin attenuates glucose uptake by inhibiting the phosphorylation of IRS in mature 3T3-L1 adipocytes. {yields} Fucoxanthin exerts its anti-obesity effect by inhibiting the differentiation of adipocytes at both intermediate and late stages, as well as glucose uptake in mature adipocytes. -- Abstract: Progression of 3T3-L1 preadipocyte differentiation is divided into early (days 0-2, D0-D2), intermediate (days 2-4, D2-D4), and late stages (day 4 onwards, D4-). In this study, we investigated the effects of fucoxanthin, isolated from the edible brown seaweed Petalonia binghamiae, on adipogenesis during the three differentiation stages of 3T3-L1 preadipocytes. When fucoxanthin was applied during the early stage of differentiation (D0-D2), it promoted 3T3-L1 adipocyte differentiation, as evidenced by increased triglyceride accumulation. At the molecular level, fucoxanthin increased protein expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), sterol regulatory element-binding protein 1c (SREBP1c), and aP2, and adiponectin mRNA expression, in a dose-dependent manner. However, it reduced the expression of PPAR{gamma}, C/EBP{alpha}, and SREBP1c during the intermediate (D2-D4) and late stages (D4-D7) of differentiation. It also inhibited the uptake of glucose in mature 3T3-L1 adipocytes by reducing the phosphorylation of insulin receptor substrate 1 (IRS-1). These results suggest that fucoxanthin exerts differing effects on 3T3-L1 cells of different differentiation stages and inhibits glucose uptake in mature adipocytes.

  9. Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways.

    PubMed

    Son, Jang-Ho; Cho, Yeong-Cheol; Sung, Iel-Yong; Kim, In-Ryoung; Park, Bong-Soo; Kim, Yong-Deok

    2014-11-01

    Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways. PMID:25250639

  10. Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells

    PubMed Central

    2012-01-01

    Background Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting. Results We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes. Conclusions We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis. PMID:22436005

  11. DNA Topoisomerase IIα contributes to the early steps of adipogenesis in 3T3-L1 cells.

    PubMed

    Jacobsen, Rhîan G; Mazloumi Gavgani, Fatemeh; Mellgren, Gunnar; Lewis, Aurélia E

    2016-10-01

    DNA topoisomerases (Topo) are multifunctional enzymes resolving DNA topological problems such as those arising during DNA replication, transcription and mitosis. Mammalian cells express 2 class II isoforms, Topoisomerases IIα (Topo IIα) and IIβ (Topo IIβ), which have similar enzymatic properties but are differently expressed, in dividing and pluripotent cells, and in post-mitotic and differentiated cells respectively. Pre-adipocytes re-enter the cell cycle prior to committing to their differentiation and we hypothesised that Topo II could contribute to these processes. We show that Topo IIα expression in 3T3-L1 cells is induced within 16h after the initiation of the differentiation programme, peaks at 24h and rapidly declines thereafter. In contrast Topo IIβ was present both in pre-adipocytes and throughout differentiation. Inhibition of PI3K with LY294002, known to prevent adipocyte differentiation, consistently reduced the expression of Topo IIα, whereas a clear effect on Topo IIβ was not apparent. In addition, inhibition of mTOR with rapamycin also reduced the protein levels of Topo IIα. Using specific class IA PI3K catalytic subunit inhibitors, we show that p110α inhibition with A66 has the greatest reduction of Topo IIα expression and of differentiation, as measured by triglyceride storage. The timing of Topo IIα expression coincides with the mitotic clonal expansion (MCE) phase of differentiation and inhibition of Topo II with ICRF-187 during this stage decreased PPARγ1 and 2 protein levels and triglyceride storage, whereas inhibition later on has little impact. Moreover, the addition of ICRF-187 had no effect on the incorporation of EdU during S-phase at day 1 but lowered the relative cell numbers on day 2. ICRF-187 also induced an increase in the centri/pericentromeric heterochromatin localisation of Topo IIα, indicating a role for Topo IIα at these locations during MCE. In summary, we present evidence that Topo IIα plays an important role

  12. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. PMID:24863237

  13. Controlled release of simvastatin from in situ forming hydrogel triggers bone formation in MC3T3-E1 cells.

    PubMed

    Park, Yoon Shin; David, Allan E; Park, Kyung Min; Lin, Chia-Ying; Than, Khoi D; Lee, Kyuri; Park, Jun Beom; Jo, Inho; Park, Ki Dong; Yang, Victor C

    2013-04-01

    Simvastatin (SIM), a drug commonly administered for the treatment of hypercholesterolemia, has been recently reported to induce bone regeneration/formation. In this study, we investigated the properties of hydrogel composed of gelatin-poly(ethylene glycol)-tyramine (GPT) as an efficient SIM delivery vehicle that can trigger osteogenic differentiation. Sustained delivery of SIM was achieved through its encapsulation in an injectable, biodegradable GPT-hydrogel. Cross-linking of the gelatin-based GPT-hydrogel was induced by the reaction of horse radish peroxidase and H(2)O(2). GPT-hydrogels of three different matrix stiffness, 1,800 (GPT-hydrogel1), 5,800 (GPT-hydrogel2), and 8,400 Pa (GPT-hydrogel3) were used. The gelation/degradation time and SIM release profiles of hydrogels loaded with two different concentrations of SIM, 1 and 3 mg/ml, were also evaluated. Maximum swelling times of GPT-hydrogel1, GPT-hydrogel2, and GPT-hydrogel3 were observed to be 6, 12, and 20 days, respectively. All GPT-hydrogels showed complete degradation within 55 days. The in vitro SIM release profiles, investigated in PBS buffer (pH 7.4) at 37°C, exhibited typical biphasic release patterns with the initial burst being more rapid with GPT-hydrogel1 compared with GPT-hydrogel3. Substantial increase in matrix metalloproteinase-13, osteocalcin expression levels, and mineralization were seen in osteogenic differentiation system using MC3T3-E1 cells cultured with GPT-hydrogels loaded with SIM in a dose-dependent manner. This study demonstrated that controlled release of SIM from a biodegradable, injectable GPT-hydrogel had a promising role for long-term treatment of chronic degenerative diseases such as disc degenerative disease. PMID:23250670

  14. Real Time Monitoring of Inhibition of Adipogenesis and Angiogenesis by (−)-Epigallocatechin-3-Gallate in 3T3-L1 Adipocytes and Human Umbilical Vein Endothelial Cells

    PubMed Central

    Tang, Wenjing; Song, Huanlei; Cai, Wei; Shen, Xiuhua

    2015-01-01

    Little is known about the effect of (−)-epigallocatechin-3-gallate (EGCG) on angiogenesis in adipocytes. We aimed to test the effect of EGCG on the expression of vascular endothelial growth factor (VEGF) in adipocytes. The levels of VEGF secretion, the expression of VEGF message ribonucleic acid (mRNA) and VEGF protein in 3T3-L1 cells were measured by enzyme linked immunosorbent assay (ELISA), real time polymerase chain reaction (PCR), and immunofluorescence staining, respectively. The xCELLigence real time cell analysis system was used to study the growth and differentiation of 3T3-L1 preadipocytes. A coculture system was used to test the effects of 3T3-L1 cells on proliferation of human umbilical vein endothelial cells (HUVECs). The conditioned media derived from 3T3-L1 cells treated with or without EGCG was used to culture the HUVECs for a tube formation assay. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), two transcription factors related to both adipogenesis and angiogenesis, were examined to explore the potential mechanism. We found that all the three measurements of VEGF expression in adipocytes (mRNA, protein and secretion in media) were reduced after EGCG treatment. The growth of HUVECs co-cultured with 3T3-L1 cells was significantly increased and the conditioned media from EGCG treated 3T3-L1 adipocytes inhibited tube formation in HUVECs. Both PPARγ and C/EBPα expression in adipocytes were decreased with EGCG treatment. In conclusion, findings from this study suggest that EGCG may inhibit angiogenesis by regulating VEGF expression and secretion in adipocytes. PMID:26516907

  15. Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells

    SciTech Connect

    Horiuchi, Shinji; Kato, Kiyoko . E-mail: kkatoh@tsurumi.beppu.kyushu-u.ac.jp; Suga, Shin; Takahashi, Akira; Ueoka, Yousuke; Arima, Takahiro; Nishida, Jun-ichi; Hachisuga, Toru; Kawarabayashi, Tatsuhiko; Wake, Norio

    2005-05-01

    Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

  16. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  17. Aspirin Breaks the Crosstalk between 3T3-L1 Adipocytes and 4T1 Breast Cancer Cells by Regulating Cytokine Production.

    PubMed

    Hsieh, Chia-Chien; Huang, Yu-Shan

    2016-01-01

    Breast cancer is one of the most common cancers in women worldwide. The obesity process is normally accompanied by chronic, low-grade inflammation. Infiltration by inflammatory cytokines and immune cells provides a favorable microenvironment for tumor growth, migration, and metastasis. Epidemiological evidence has shown that aspirin is an effective agent against several types of cancer. The aim of this study is to investigate the anti-inflammatory and anti-cancer effects of aspirin on 3T3-L1 adipocytes, 4T1 murine breast cancer cells, and their crosstalk. The results showed that aspirin treatment inhibited differentiation and lipid accumulation by 3T3-L1 preadipocytes, and decreased the secretion of the inflammatory adipokine MCP-1 after stimulation with tumor necrosis factor (TNF)-α or conditioned medium from RAW264.7 cells. In 4T1 cells, treatment with aspirin decreased cell viability and migration, possibly by suppressing MCP-1 and VEGF secretion. Subsequently, culture of 4T1 cells in 3T3-L1 adipocyte-conditioned medium (Ad-CM) and co-culture of 3T3-L1 and 4T1 cells using a transwell plate were performed to clarify the relationship between these two cell lines. Aspirin exerted its inhibitory effects in the transwell co-culture system, as well as the conditioned-medium model. Aspirin treatment significantly inhibited the proliferation of 4T1 cells, and decreased the production of MCP-1 and PAI-1 in both the Ad-CM model and co-culture system. Aspirin inhibited inflammatory MCP-1 adipokine production by 3T3-L1 adipocytes and the cell growth and migration of 4T1 cells. It also broke the crosstalk between these two cell lines, possibly contributing to its chemopreventive properties in breast cancer. This is the first report that aspirin's chemopreventive activity supports the potential application in auxiliary therapy against obesity-related breast cancer development. PMID:26794215

  18. GPER mediates the inhibitory actions of estrogen on adipogenesis in 3T3-L1 cells through perturbation of mitotic clonal expansion.

    PubMed

    Zhu, Pei; Yuen, Jacky M L; Sham, Kathy W Y; Cheng, Christopher H K

    2013-11-01

    G-protein-coupled estrogen receptor 1 (GPER) mediates non-genomic signaling of estrogenic events. Here we showed for the first time that Gper/GPER is expressed in Swiss 3T3 mouse embryo preadipocytes 3T3-L1, and that Gper/GPER is up-regulated during differentiation of the cells induced by monocyte differentiation-inducing (MDI) cocktail. Activation of GPER by the natural ligand 17β-estradiol (E2), and the specific agonist G1, was shown to inhibit lipid accumulation in 3T3-L1 cells, while such inhibition was reversed upon knockdown of GPER using specific siRNA. GPER was also found to mediate perturbation of mitotic clonal expansion (MCE) in these cells by inhibiting cell cycle arrest during MDI cocktail-induced differentiation. Persistent activation of cell cycle regulating factors cyclin-dependant kinase (CDK) 4, CDK6 and cyclin D1, and phosphorylation of retinoblastoma (Rb) protein at serine 795 was observed in the G1-treated cells. Taken together, our results indicate that E2-GPER signaling leads to an inhibition of adipogenesis in 3T3-L1 cells via perturbation of MCE. PMID:23871778

  19. Cytotoxicity of folic acid conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells, with and without encapsulated DOX.

    PubMed

    Patel, Kunal; Sundara Raj, Behin; Chen, Yan; Lou, Xia

    2016-04-01

    Hollow silica nanoparticles of two sizes with and without a folic acid targeting ligand were synthesized. Fickian diffusion of the antitumor drug doxorubicin hydrochloride (DOX) was demonstrated by the produced nanoparticles, achieving a cumulative release of 73% and 45% for 215 nm and 430 nm particles respectively over a period of 500 h. The hollow silica nanoparticles presented a time and dose dependent toxicity, selective to human epithelial colorectal adenocarcinoma (Caco2) cells, over mouse embryonic fibroblast (3T3) cells. At 24h Caco2 cell viability was reduced to 66% using pure hollow silica at a concentration of 50 μg mL(-1), while that of 3T3 cells remained at 94% under the same conditions. The selective cytotoxicity of hollow silica nanoparticles was further enhanced by conjugation of folic acid and incorporation of DOX: at 24h and an equivalent DOX concentration of 0.5 μg mL(-1), viable Caco2 cells were reduced to 45% while 3T3 cells were reduced to 83%. Interestingly the equivalent dose of free DOX was more toxic to 3T3 than to Caco2 cells, reducing the 3T3 viability to 72% and the Caco2 viability to 80%, which is likely due to the presence of the p-glycoprotein pumps in Caco2 cells. Folic acid conjugation served to enhance the viability of both cell lines in this work. Careful optimization of the folate content should further improve the cell specificity of the hollow silica nanoparticles, thus providing a viable targeting platform for cancer therapy. PMID:26764104

  20. Chemopreventive effect of punicalagin, a novel tannin component isolated from Terminalia catappa, on H-ras-transformed NIH3T3 cells.

    PubMed

    Chen, Pin-Shern; Li, Jih-Heng

    2006-05-01

    Terminalia catappa and its major tannin component, punicalagin, have been characterized to possess antioxidative and anti-genotoxic activities. However, their effects on reactive oxygen species (ROS) mediated carcinogenesis are still unclear. In the present study, H-ras-transformed NIH3T3 cells were used to evaluate the chemopreventive effect of T. catappa water extract (TCE) and punicalagin. In the cell proliferation assay, TCE and punicalagin suppressed the proliferation of H-ras-transformed NIH3T3 cells with a dose-dependent manner but only partially affected non-transformed NIH3T3 cells proliferation. The differential cytotoxicity of TCE/punicalagin on the H-ras-transformed and non-transformed NIH3T3 cells indicated the selectivity of TCE/punicalagin against H-ras induced transformation. TCE or punicalagin treatment reduced anchorage-independent growth that could be due to a cell cycle arrest at G0/G1 phase. The intracellular superoxide level, known to modulate downstream signaling of Ras protein, was decreased by punicalagin treatments. The levels of phosphorylated JNK-1 and p38 were also decreased with punicalagin treatments. Thus, the chemopreventive effect of punicalagin against H-ras induced transformation could result from inhibition of the intracellular redox status and JNK-1/p38 activation. PMID:16242868

  1. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line.

    PubMed

    Śmieszek, Agnieszka; Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marędziak, Monika; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  2. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    PubMed Central

    Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  3. Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system.

    PubMed

    Kramer, Adam H; Joos-Vandewalle, Julia; Edkins, Adrienne L; Frost, Carminita L; Prinsloo, Earl

    2014-01-24

    Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors. PMID:24388983

  4. Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.

    PubMed Central

    Egan, S E; McClarty, G A; Jarolim, L; Wright, J A; Spiro, I; Hager, G; Greenberg, A H

    1987-01-01

    Using three independent approaches, we studied the effects of H-ras on metastasis formation. Analysis of five in vitro-ras-transfected 10T1/2 clones with either flat or refractile morphologies revealed a relationship between metastatic potential, H-ras expression, and anchorage-independent growth. Four metastatic variants derived from a poorly metastatic, low-H-ras-expressing line all expressed high levels of H-ras RNA and grew efficiently in soft agar. Activation of H-ras expression in the metastatic tumors had occurred through amplification and rearrangement of H-ras sequences. In addition, preinduction of p21 synthesis in NIH 3T3 line 433, which contains v-H-ras under transcriptional control of the glucocorticoid-sensitive mouse mammary tumor virus long terminal repeat, significantly increased metastatic efficiency. Glucocorticoid treatment of normal or pEJ-transformed NIH 3T3 cells did not affect metastatic potential. These data reveal a direct relationship between ras expression and metastasis formation and suggest that metastatic and transformed phenotypes may be coregulated in ras-transformed 10T1/2 and NIH 3T3 cells. Images PMID:3102946

  5. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    SciTech Connect

    An, Lei; Pang, Yun-Wei; Gao, Hong-Mei; Tao, Li; Miao, Kai; Wu, Zhong-Hong; and others

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. Black-Right-Pointing-Pointer fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. Black-Right-Pointing-Pointer fat-1 reduces lipid deposition in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  6. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells.

    PubMed

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-09-01

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ-induced activity and expression of adipocyte-specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1-PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of

  7. Effect of Thymosin beta4 on the Differentiation and Mineralization of MC3T3-E1 Cell on a Titanium Surface.

    PubMed

    Jeong, Soon-Jeong; Jeong, Moon-Jin

    2016-02-01

    Osteoblasts are responsible for the synthesis of bone matrix through the secretion of collagenous and non-collagenous proteins with mineralization. Thymosin beta4 (Tbeta4) is an actin-sequestering peptide that is involved in the regulation of cell proliferation, differentiation and motility. A recent study reported that the inhibition of Tbeta4 mRNA synthesis strongly decreases the level of gene expression of bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), osteocalcin (OCN), osteonectin (ON) and collagen type I (Col I) with mineralization during differentiation in odontoblasts. Titanium (Ti) is used commonly as an implant material for dental implants, which have strong mechanical potential and good biocompatibility with bone. This study examined whether Tbeta4 can be a potential molecule for promoting the differentiation and mineralization of MC3T3-E1 cells on a Ti surface. Tbeta4 increased the viability of MC3T3-E1 cells during differentiation on Ti discs compared to that of the control. The expression of Tbeta4 mRNA and protein in the Tbeta4-treated MC3T3-E1 cells was higher than the control during differentiation on the Ti discs. In addition, Tbeta4 increased the formation of mineralization nodules and the mRNA expression of alkaline phosphatase (ALP), DSPP, dentin matrix protein1 (DMP1), BSP and Col I compared to that of the control in MC3T3-E1 cells during differentiation on Ti discs. From the results, Tbeta4 increased the viability and promoted the differentiation and mineralization of MC3T3-E1 cells on Ti discs. This highlights the potential use of Tbeta4 for increasing osseointegration through osteoblast differentiation and mineralization on Ti discs. PMID:27433712

  8. Effects of large and small T antigens on DNA synthesis and cell division in simian virus 40-transformed BALB/c 3T3 cells.

    PubMed Central

    Christensen, J B; Brockman, W W

    1982-01-01

    The roles of the large T and small t antigens of simian virus 40 in cellular DNA synthesis and cell division were analyzed in BALB/c 3T3 mouse cells transformed by wild-type, temperature-sensitive A (tsA), or tsA-deletion (tsA/dl) double mutants. Assessment of DNA replication and cell cycle distribution by radioautography of [3H]thymidine-labeled nuclei and by flow microfluorimetry indicate that tsA transformants do not synthesize DNA or divide at the restrictive temperature to the same extent as they do at the permissive temperature or as wild-type transformants do at the restrictive temperature. This confirms earlier studies suggesting that large T induces DNA synthesis and mitosis in transformed cells. Inhibition of replication in tsA transformants at the restrictive temperature, however, is not complete. Some residual cell division does occur but is in large part offset by cell detachment and death. This failure to revert completely to the parental 3T3 phenotype, as indicated by residual cell cycling at the restrictive temperature, was also observed in cells transformed by tsA/dl double mutants which, in addition to producing a ts large T, make no small t protein. Small t, therefore, does not appear to be responsible for the residual cell cycling and plays no demonstrable role in the induction of DNA synthesis or cell division in stably transformed BALB/c 3T3 cells. Comparison of cell cycling in tsA and tsA/dl transformants, normal 3T3 cells, and a transformation revertant suggests that the failure of tsA transformants to revert completely may be due to leakiness of the tsA mutation as well as to a permanent cellular alteration induced during viral transformation. Finally, analysis of cells transformed by tsA/dl double mutants indicates that small t is not required for full expression of growth properties characteristic of transformed cells. Images PMID:6292518

  9. The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.

    PubMed

    Nagamoto, Yasuhito; Tashiro, Katsuhisa; Takayama, Kazuo; Ohashi, Kazuo; Kawabata, Kenji; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

    2012-06-01

    Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. PMID:22445253

  10. Actions of β-Apo-Carotenoids in Differentiating Cells: Differential Effects in P19 Cells and 3T3-L1 Adipocytes

    PubMed Central

    Wang, Cynthia X.; Jiang, Hongfeng; Yuen, Jason J.; Lee, Seung-Ah; Narayanasamy, Sureshbabu; Curley, Robert W.; Harrison, Earl H.; Blaner, William S.

    2015-01-01

    β-Apo-carotenoids, including β-apo-13-carotenone and β-apo-14′-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14′-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10′-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation. PMID:25602703

  11. Delivering MC3T3-E1 cells into injectable calcium phosphate cement through alginate-chitosan microcapsules for bone tissue engineering*

    PubMed Central

    Qiao, Peng-yan; Li, Fang-fang; Dong, Li-min; Xu, Tao; Xie, Qiu-fei

    2014-01-01

    Objective: To deliver cells deep into injectable calcium phosphate cement (CPC) through alginate-chitosan (AC) microcapsules and investigate the biological behavior of the cells released from microcapsules into the CPC. Methods: Mouse osteoblastic MC3T3-E1 cells were embedded in alginate and AC microcapsules using an electrostatic droplet generator. The two types of cell-encapsulating microcapsules were then mixed with a CPC paste. MC3T3-E1 cell viability was investigated using a Wst-8 kit, and osteogenic differentiation was demonstrated by an alkaline phosphatase (ALP) activity assay. Cell attachment in CPC was observed by an environment scanning electron microscopy. Results: Both alginate and AC microcapsules were able to release the encapsulated MC3T3-E1 cells when mixed with CPC paste. The released cells attached to the setting CPC scaffolds, survived, differentiated, and formed mineralized nodules. Cells grew in the pores concomitantly created by the AC microcapsules in situ within the CPC. At Day 21, cellular ALP activity in the AC group was approximately four times that at Day 7 and exceeded that of the alginate microcapsule group (P<0.05). Pores formed by the AC microcapsules had a diameter of several hundred microns and were spherical compared with those formed by alginate microcapsules. Conclusions: AC microcapsule is a promising carrier to release seeding cells deep into an injectable CPC scaffold for bone engineering. PMID:24711359

  12. IL-1α induces apoptosis and inhibits the osteoblast differentiation of MC3T3-E1 cells through the JNK and p38 MAPK pathways.

    PubMed

    Guo, Chun; Yang, Xu-Guang; Wang, Fei; Ma, Xu-Yuan

    2016-07-01

    Interleukin (IL)-1 is a proinflammatory cytokine that plays important roles in inflammation and host responses to infection. The present study aimed to evaluate the effects of IL-1α on the apoptosis and differentiation of osteoblasts, and to elucidate the mechanism responsible for these effects in the osteoblast‑like cell line MC3T3-E1. The MC3T3-E1 cells were non-treated or treated with IL-1α. Following treatment, cell viability, alkaline phosphatase (ALP) activity and caspase-3 activity were evaluated. The expression of osteoblast-specific genes as well as Bax, Bcl-2 and caspase-3 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of Bax, Bcl-2, caspase-3 and the phosphorylation of mitogen-activated protein kinases (MAPKs, also known as MAP kinases) were evaluated using western blot analysis. The MAPK signaling pathway was blocked by pre-treatment with MAPK inhibitors SB203580, PD98059 and SP600125. IL-1α treatment induced a significant decrease in cell viability and ALP activity in the MC3T3-E1 cells. IL-1α also significantly decreased the mRNA expression and protein levels of osteoblast-related genes in the MC3T3-E1 cells. On the other hand, IL-1α significantly upregulated the mRNA expression and protein levels of Bax and caspase-3 as well as caspase-3 activity, whereas Bcl-2 expression was decreased in the MC3T3-E1 cells. Furthermore, IL-1α activated the apoptotic signaling pathway by increasing the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-MAPK, whereas it inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, pre-treatment with MAPK inhibitors attenuated the phosphorylation of JNK, p38 and Bax expression enhanced by IL-1α. However, MAPK inhibitors markedly increased the protein expression of osteoblast-related genes and Bcl-xL in the MC3T3-E1 cells downregulated by IL-1α. Taken together, these findings suggest that IL-1α induces

  13. Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells.

    PubMed

    Satué, María; Arriero, Maria del Mar; Monjo, Marta; Ramis, Joana Maria

    2013-11-15

    Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48h of treatment, doses ≥100μM of diosmetin and galangin and also 500μM taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ≥100μM of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500μM) and taxifolin (100 and 200μM) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100μM taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism. PMID:24060614

  14. Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca2+-sensitive phospholipid-dependent protein kinase.

    PubMed Central

    Rozengurt, E; Rodriguez-Pena, A; Coombs, M; Sinnett-Smith, J

    1984-01-01

    The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol competes directly with [3H]phorbol 12,13-dibutyrate for common binding sites in monolayer cultures of Swiss 3T3 cells and rapidly stimulates the phosphorylation of a Mr 80,000 cellular protein that has recently been shown to reflect the activation of protein kinase C in intact cells. Thus, this diacylglycerol provided a useful tool to determine whether exogenously added diacylglycerols can mimic the potent tumor promoter phorbol ester in eliciting DNA synthesis and cell division in quiescent cells. We found that OAG acts synergistically with insulin and other growth factors to stimulate reinitiation of cell proliferation, and several lines of evidence indicate that OAG shares with phorbol esters a common pathway of mitogenic action via stimulation of protein kinase C activity in intact 3T3 cells. The findings support the hypothesis that diacylglycerols represent endogenous analogs of phorbol esters and raise the possibility that diacylglycerols generated in the plasma membrane could act as a mitogenic signal for quiescent cells. Images PMID:6237364

  15. A quantitative study of MC3T3-E1 cell adhesion, morphology and biomechanics on chitosan-collagen blend films at single cell level.

    PubMed

    Wang, Chuang; Xie, Xu-dong; Huang, Xun; Liang, Zhi-hong; Zhou, Chang-ren

    2015-08-01

    The interaction between cells and biomaterials plays a key role in cell proliferation and differentiation in tissue engineering. However, a quantitative analysis of those interactions has been less well studied. The objective of this study was to quantitative recapitulate the difference of MC3T3-E1 cell adhesion, morphological and biomechanical properties on chitosan-collagen films in terms of chemical composition. Here, the unbinding force between MC3T3-E1 cell and a series of chitosan-collagen films was probed by a real-time and in situ atomic force microscopy-single cell force spectroscopy (AFM-SCFS). Meanwhile, changes in cell morphology and Young's modulus on different chitosan-collagen films were detected by AFM. The cell area and CCK-8 results showed that cell spreading and proliferation increased with increasing collagen content. AFM observations clearly showed cell height decreased and pseudopod fusion with the collagen content increased. Cell adhesive force increased from 0.76±0.17 nN to 1.70±0.19 nN. On the contrary, cells Young's modulus, which reflected biophysical changes of cells decreased from 11.94±3.19 kPa to 1.81±0.52 kPa, respectively. It suggested that stronger cell-substrate interactions benefit cell adhesion, and better cell flexibility improve cell spreading. The findings indicate that cell morphology, adhesive force and Young's modulus are significant affected by various chitosan-collagen substrates. Those methods and quantitative results have guiding significance for investigating the mechanism of chitosan and/or collagen based cell-targeting drug carrier and the preparation of chitosan-collagen composite biomaterials. PMID:25996415

  16. Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

    PubMed Central

    Kawamura, M; Jensen, D F; Wancewicz, E V; Joy, L L; Khoo, J C; Steinberg, D

    1981-01-01

    Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation. PMID:6262767

  17. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    PubMed Central

    Zhong, Xing; Xiu, Ling-ling; Wei, Guo-hong; Liu, Yuan-yuan; Su, Lei; Cao, Xiao-pei; Li, Yan-bing; Xiao, Hai-peng

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects. Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins. Results: Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation. Conclusion: Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation. PMID:21499286

  18. Effects of modified Shu-Gan-Liang-Xue decoction combined with anastrozole on osteoblastic proliferation and differentiation of MC3T3-E1 cells

    PubMed Central

    ZHOU, FEI; HAN, SHUYAN; ZHOU, NING; ZHENG, WENXIAN; LI, PINGPING

    2015-01-01

    Aromatase inhibitors (AIs) are widely used in the treatment of hormone-dependent breast cancer and as a result, aromatase inhibitor-associated bone loss (AIBL) has become a major concern amongst patients receiving AI treatment. Modified Shu-Gan-Liang-Xue decoction (mSGLXD), a clinical prescription, has been used for ameliorating AIBL in patients with breast cancer for decades and has achieved good clinical efficacy. However, the mechanism underlying how mSGLXD influences bone homeostasis and alleviates AIBL has remained elusive. In the present study, mSGLXD was supplemented with Rhizoma Drynariae containing phytoestrogens, and the safety of mSGLXD was evaluated. mSGLXD did not possess estrogenic activity and significantly inhibited the proliferation of estrogen receptor-positive breast cancer cell line MCF-7, which suggested that mSGLXD was safe for postmenopausal patients with breast cancer. Subsequently, the effects of mSGLXD alone or in combination with anastrozole on osteoblastic MC3T3-E1 cell proliferation and differentiation were investigated. Cell counting kit-8, reverse transcription-polymerase chain reaction and biochemical methods, such as ELISA and alizarin red S staining, were used in the present study. It was revealed that mSGLXD not only stimulated MC3T3-E1 cell proliferation, but also upregulated alkaline phosphatase and osteocalcin gene and protein expression levels. High concentrations of anastrozole (10 or 100 μmol/l) markedly inhibited MC3T3-E1 cell proliferation, but this inhibitory effect was attenuated by mSGLXD. Furthermore, mSGLXD increased MC3T3-E1 cell mineralization following β-glycerophosphate and ascorbic acid induction. Therefore, the results of the present study suggested that mSGLXD may be a promising adjuvant therapy, with high safety and efficacy, for the prevention and treatment of AIBL in patients with breast cancer who receive AI treatment. PMID:25405542

  19. Inhibitory effects of compounds isolated from the dried branches and leaves of murta (Myrceugenia euosma) on lipid accumulation in 3T3-L1 cells.

    PubMed

    Oikawa, Naoki; Nobushi, Yasuhito; Wada, Taira; Sonoda, Kumiko; Okazaki, Yuzo; Tsutsumi, Shigetoshi; Park, Yong Kun; Kurokawa, Masahiko; Shimba, Shigeki; Yasukawa, Ken

    2016-07-01

    As obesity is a global health concern the demand for anti-obesity drugs is high. In this study, we investigated the anti-obesity effect of the dried branches and leaves of murta (Myrceugenia euosma Legrand, Myrtaceae). A methanol extract of the dried branches and leaves of murta inhibited adipogenesis in 3T3-L1 cells. Three known flavanones-cryptostrobin (1), pinocembrin (4), and 5,7-dihydroxy-6,8-dimethylflavanone (6), and three chalcones-2',6'-dihydroxy-3'-methyl-4'-methoxychalcone (2), pinostrobin chalcone (3), and 2',6'-dihydroxy-4'-methoxy-3',5'-dimethylchalcone (5) were isolated from the active fraction. Structures of these compounds were identified using various spectral data. Each of these compounds also inhibited adipogenesis in 3T3-L1 cells. In particular, compound 3 was a more potent inhibitor of triglyceride accumulation than the positive control berberine. Gene expression studies revealed that treatment of 3T3-L1 cells with 3 lowers the expression levels of CCAAT/enhancer-binding protein α and peroxisome proliferator activator γ2 during adipogenesis without affecting cell viability. Treatment of 3T3-L1 cells with 3 reduced the expression levels of mRNAs encoding sterol regulatory element-binding protein 1c and several lipogenic enzymes, including fatty acid synthase and stearoyl CoA desaturase-1. These results indicate that the methanol extract and compounds isolated from the dried branches and leaves of murta exert their anti-obesity effects through the inhibition of adipogenesis. PMID:26880616

  20. Relationship between exposure to TPA and appearance of transformed cells in MNNG-initiated transformation of BALB/c 3T3 cells.

    PubMed

    Tsuchiya, T; Umeda, M

    1997-10-01

    In the BALB/c-3T3-cell transformation system, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure on the appearance of transformed cells was examined in order to investigate the mechanisms of in vitro tumor promotion. Optimal duration of TPA exposure on N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cells was at least 11 days. To investigate the effect of transformation frequencies of altering inoculating cell density at the replating of MNNG-exposed cells and of altering the time of starting TPA exposure, MNNG-exposed cells were replated at various inoculum sizes. With lower inoculum sizes (1 x 10(3) to 3 x 10(4) cells/dish), maximum TPA-induced transformation occurred for TPA commencement at confluence, while with higher inoculum size (1 x 10(5) cells/dish), maximum transformation frequency was observed when TPA exposure was started on day 7 after replating, being some 2 days after confluence. This may suggest that there are different mechanisms involved, depending on inoculum size, and that these may involve cell-cell interactions (at lower inoculum) and mutation expression periods (at higher inoculum). By means of redispersion experiments, it was demonstrated that the appearance of transformed cells begins on about day 7 after replating at a cell density of 1 x 10(4) cells/dish. These results suggest the usefulness of the replating method for optimizing transformation in the BALB/c-3T3-cell transformation assay, and provide insight into the time frame of expression of MNNG-initiated transformants and TPA-induced expansion of these transformants. PMID:9335454

  1. Effect of Fermented Red Ginseng Extract Enriched in Ginsenoside Rg3 on the Differentiation and Mineralization of Preosteoblastic MC3T3-E1 Cells.

    PubMed

    Siddiqi, Muhammad Zubair; Siddiqi, Muhammad Hanif; Kim, Yeon-Ju; Jin, Yan; Huq, Md Amdadul; Yang, Deok-Chun

    2015-05-01

    In this study, red ginseng extract (RGE) was converted into high-content minor ginsenosides by fermenting with Bgp1 enzymes at 37°C for 5 days. Compared to the RGE, the minor ginsenoside contents were increased in fermented red ginseng extract (FRGE). Moreover, the amount of minor ginsenosides such as Rh1 (11%) and Rg2 (16%) was slightly augmented, while the level of Rg3 (33%) was significantly increased after bioconversion. Furthermore, we also examined and compared the effect of RGE and FRGE on the differentiation and mineralization of preosteoblastic MC3T3-E1 cells. Similarly, the level of mRNA expression of intracellular alkaline phosphatase (ALP) activity, type-1 collagen (Col-I) was also increased. Based on the comparison, it is clear that the FRGE has improved effects on bone formation and differentiation of preosteoblastic MC3T3-E1 cells. PMID:25764149

  2. Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

    SciTech Connect

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1986-05-01

    Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/sup 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.

  3. Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

    PubMed

    Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

    2012-11-01

    This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3. PMID:23066647

  4. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe3O4 nanofibers with static magnetic field exposure.

    PubMed

    Cai, Qing; Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun; Deng, Xuliang; Yang, Xiaoping

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT-1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. PMID:26117751

  5. The β-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1

    PubMed Central

    Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

    2014-01-01

    Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (β-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. β-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of β-SiC nanowires was investigated for the first time. Our results indicated that 100 nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100 μm long SiC nanowires. And 100 nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100 nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, β-SiC nanowires may have limitations as medical material. PMID:24967352

  6. Ginsenoside F2 possesses anti-obesity activity via binding with PPARγ and inhibiting adipocyte differentiation in the 3T3-L1 cell line.

    PubMed

    Siraj, Fayeza Md; SathishKumar, Natarajan; Kim, Yeon Ju; Kim, Se Young; Yang, Deok Chun

    2015-02-01

    Abstract Panax ginseng Meyer has been shown to be effective in mitigating various diseases. Protopanaxadiols (PPD) and protopanaxatriols (PPT), which are the main constituents of ginseng, have been shown to impact obesity. Therefore, we selected several important ginsenosides to perform our docking study and determine if they had binding affinity with the peroxisome proliferator activated receptor gamma (PPARγ), which is a major transcription factor in adipocytes. Among them, only a few ginsenosides demonstrated binding affinity with PPARγ. Other than ginsenoside F2 rest of them were previously reported by the researchers in experimental study in case of obesity cell line 3T3-L1 adipocyte. In few recent studies, it was reported that F2 has protective effects on malignant brain tumors as well as anti-cancer activity in breast cancer. Therefore, we felt it was important to focus on F2 when considering obesity. Our study focused on this ginsenoside and analyzed its impact on 3T3-L1 adipocytes. Following the molecular interaction studies, further experimental studies were carried out and demonstrated that ginsenoside F2 when treated with different doses reduces the level of lipid accumulated by the 3T3-L1 cell line during adipogenesis. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR results showed reduction in PPARγ and perilipin gene expression levels compared to that of differentiated adipocytes without any treatment. So considering the binding with a major adipocyte transcription factor and the performed experiments, we suggest that ginsenoside F2 may reduce obesity via the inhibition of adipogenesis in the 3T3-L1 cell line. PMID:24666293

  7. Quercetin reversed lipopolysaccharide-induced inhibition of osteoblast differentiation through the mitogen‑activated protein kinase pathway in MC3T3-E1 cells.

    PubMed

    Wang, Xin-Chun; Zhao, Nzhi-Jun; Guo, Chun; Chen, Jing-Tao; Song, Jin-Ling; Gao, Li

    2014-12-01

    Quercetin, a flavonoid found in onions and other vegetables, has potential inhibitory effects on bone resorption in vivo and in vitro. In our previous study it was identified that quercetin triggered the apoptosis of lipopolysaccharide (LPS)‑induced osteoclasts and inhibited bone resorption. Currently, little information is available detailing the effect of quercetin on osteoblast differentiation and bone formation in bacteria‑induced inflammatory diseases. The present study aimed to investigate the effect of quercetin on osteoblast differentiation in MC3T3‑E1 osteoblasts stimulated with LPS. LPS significantly downregulated the mRNA expression of osteoblast‑related genes in the MC3T3‑E1 cells. By contrast, quercetin significantly restored the LPS‑suppressed mRNA expression of osteoblast‑related genes in a dose‑dependent manner. Quercetin also restored the protein expression of Osterix in MC3T3‑E1 cells suppressed by LPS. Furthermore, quercetin selectively triggered the activation of the mitogen‑activated protein kinase (MAPK) pathway by enhancing the expression of extracellular signal-regulated kinase and reducing the expression of c‑Jun N‑terminal kinase. These data suggest that quercetin reversed the inhibition of osteoblast differentiation induced by LPS through MAPK signaling. These findings suggest that quercetin may be of potential use as a therapeutic agent to restore osteoblast function in bacteria‑induced bone diseases. PMID:25323558

  8. Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells.

    PubMed

    Maldonado-Cervantes, Enrique; Jeong, Hyung Jin; León-Galván, Fabiola; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; González de Mejia, Elvira; de Lumen, Ben O; Barba de la Rosa, Ana P

    2010-09-01

    Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H(3) and H(4) in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits. PMID:20599579

  9. A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor

    SciTech Connect

    Hartzell, S.; Ryder, K.; Lanahan, A.; Nathans, D.; Lau, L.F.

    1989-06-01

    Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. The authors report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. The authors' results raise the possibility that mTF may also play a role in cell growth.

  10. Effect of quinupristin/dalfopristin on 3T3 and Eahy926 cells in vitro in comparison to other antimicrobial agents with the potential to induce infusion phlebitis.

    PubMed

    Kruse, Matthias; Kilic, Bülent; Flick, Burkhard; Stahlmann, Ralf

    2007-06-01

    Infusion phlebitis is a common clinical problem that is observed with some antimicrobial agents, when being administered intravenously. In this study, cultured murine fibroblasts and immortalised human endothelial cells were exposed to three antibiotics at clinically relevant concentrations to assess their toxic potential in two established cytotoxicity assays. BALB/c 3T3 fibroblasts and Eahy926 endothelial cells were exposed to quinupristin/dalfopristin (QD), erythromycin and levofloxacin at increasing concentrations. For assessment of cytotoxicity the cells were incubated with neutral red (NR) or stained with crystal violet (CV). Measurements were done by photometry. At the concentration range tested QD and erythromycin showed a concentration-dependent cytotoxic effect in both cell cultures. In 3T3 cells the half-maximal effect concentration (EC50) was 20 mg/l for QD and 340 mg/l for erythromycin in the NR uptake test and 12 and 200 mg/l, respectively, in the CV assay. In Eahy926 cells the EC50 was 50 mg/l for QD and 880 mg/l for erythromycin in the NR uptake test and 40 and 750 mg/l, respectively, in the CV assay. No EC50 could be established in both cell types for levofloxacin. Eahy926 cells were less sensitive to cytotoxic stimuli than 3T3 fibroblasts. Cytotoxic effects in both cell cultures occurred in the following order: QD > erythromycin > levofloxacin. This ranking correlates well with the frequency of local adverse effects observed with the infusion of these antibiotics in patients. Thus, these in vitro assays may serve as an estimate for the prediction of local tolerability of antibiotics when administered parenterally. PMID:17119926

  11. Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

    SciTech Connect

    Miller, D.R.; Hamby, K.M.; Slaga, T.J.

    1982-07-01

    Mouse epidermal cells can be subcultured at 31/sup 0/C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.

  12. Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity

    PubMed Central

    Jang, Sun-Hee; Lee, Soo-Jung; Ko, Yeoung-Gyu; Kim, Gon-Sup; Cho, Jae-Hyeon

    2013-01-01

    This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat

  13. Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression.

    PubMed

    Shang, Wenbin; Yang, Ying; Jiang, Boren; Jin, Hua; Zhou, Libin; Liu, Shangquan; Chen, Mingdao

    2007-01-23

    Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis. PMID:17129589

  14. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release.

    PubMed

    Yao, Qingqing; Li, Wei; Yu, Shanshan; Ma, Liwei; Jin, Dayong; Boccaccini, Aldo R; Liu, Yong

    2015-11-01

    Novel chitosan-polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. PMID:26249617

  15. Zinc deficiency induced in Swiss 3T3 cells by a low-zinc medium impairs calcium entry and two mechanisms of entry are involved.

    PubMed

    O'Dell, Boyd L; Browning, Jimmy D

    2013-04-01

    Zinc deficiency in 3T3 cells induced by the use of diethylenetriaminepentaacetate (DTPA) has been shown to impair calcium entry associated with failure of proliferation when the cells are stimulated with polypeptide growth factors (GF). These functions of zinc have been evaluated here in the same clone of cells by simple depletion using a low-zinc medium (0.05 μmol/L zinc) without chelator. Confluent cells were maintained for 1 day in the low-zinc medium without GF, then loaded with Fluo-4, and stimulated with GF. Calcium entry was measured by the increase in sustained fluorescence. It was preceded by the release of stored calcium as observed in the previous study using DTPA. Zinc deprivation decreased calcium entry when calcium was added at 0 or 0.05 mmol/L but not when 0.1 mmol/L or higher. Cell proliferation reflected similar effects of zinc and calcium concentrations. In a newly acquired clone of 3T3 cells, GF did not induce internal calcium release but thapsigargin (TG) did. When added in a low-calcium medium, both agonists stimulated calcium entry when external calcium was added, suggesting that two different mechanisms of entry were impaired by zinc deficiency. Zinc deficiency produced by DTPA in the newer clones gave similar results, decreasing calcium entry induced by both agonists. The effects of GF and TG were not additive. The results confirm the earlier observation that zinc deficiency impairs calcium entry into 3T3 cells when stimulated by GF and show that the cells can take up calcium by either store-operated or receptor-operated mechanisms. PMID:23292302

  16. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    SciTech Connect

    Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. )

    1990-06-01

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

  17. Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

    PubMed

    Faucheux, N; Nagel, M D

    2002-06-01

    Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling. PMID:12013176

  18. Substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro

    SciTech Connect

    Woll, P.J.; Rozengurt, E. )

    1988-03-01

    In the search for a more potent bombesin antagonist, the authors found (D-Arg{sup 1},D-Phe{sup 5},D-Trp{sup 7,9},Leu{sup 11})substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and other bombesin-like peptides; (ii) inhibition of {sup 125}I-labeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the M{sub r} 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis-calcium mobilization and inhibition of epidermal growth factor binding. Substance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications.

  19. Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wang, Jin-Ning; Pi, Bin; Wang, Peng; Li, Xue-Feng; Yang, Hui-Lin; Zhu, Xue-Song

    2015-09-01

    The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/α-tricalcium phosphate composite (Sema3A CMs/SF/α-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/α-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/α-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/α-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/α-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/α-TCP and SF/α-TCP composites. In conclusion, Sema3A CMs/SF/α-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

  20. Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys.

    PubMed

    Bueno-Vera, J A; Torres-Zapata, I; Sundaram, P A; Diffoot-Carlo, N; Vega-Olivencia, C A

    2015-12-01

    Electrochemical impedance spectroscopy (EIS) was used to study the behavior of MC3T3-E1 cells cultured in an αMEM+FBS solution on two Ti-based alloys (Ti-6Al-4V and γTiAl) for 4, 7 and 14 days. EIS measurements were carried out at an open-circuit potential in a 1 mHz to 100 kHz frequency range. Results indicate a general increase in impedance on the Ti alloy surfaces with cells as a function of time. Bode plots indicate changes corresponding to the passive oxide film, adsorption of proteins and cell tissue on surfaces with the passage of time. Normal cellular activity based on the polygonal morphology, with long and fine cytoplasmic prolongations of the cells on Ti-6Al-4V and γTiAl was observed from SEM images. Similarly, mineralization nodules corresponding to cell differentiation associated with the osseogenetic process were observed confirmed by Alizarin Red S staining. Immunofluorescence analysis to detect the presence of collagen Type I showed an increase in the segregation of collagen as a function of time. The impedance values obtained from EIS testing are indicative of the corrosion protection offered to the Ti alloy substrates by the cell layer. This study shows that γTiAl has better corrosion resistance than that of Ti-6Al-4V in the αMEM+FBS environment in the presence of MC3T3-E1 cells. PMID:26145813

  1. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  2. Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase C-depleted Swiss 3T3 cells.

    PubMed Central

    Corps, A N; Cheek, T R; Moreton, R B; Berridge, M J; Brown, K D

    1989-01-01

    Single-cell fluorescence image analysis has been used to characterize the mitogen-induced increases in intracellular free [Ca2+] ([Ca2+]i) in control and protein kinase C-depleted Swiss 3T3 cells. More than 80% of the control cells exhibited fast, transient responses to bombesin, vasopressin, or prostaglandin F2 alpha (PGF2 alpha). In contrast, the [Ca2+]i responses induced by platelet-derived growth factor (PDGF) were markedly more heterogeneous, slower, and often biphasic, with fewer cells (60-70%) responding. The peak [Ca2+]i values obtained in response to each mitogen showed substantial variation between cells. Brief pretreatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) reduced the [Ca2+]i responses to bombesin, but did not affect the responses to PDGF. Long-term pretreatment of the cells with TPA to down-modulate protein kinase C resulted in substantially prolonged [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha, but had no such effect on the responses to PDGF. We conclude that differences between the [Ca2+]i responses to bombesin and PDGF, previously reported using cell populations, reflect differences occurring in individual cells, and that the [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha (but not PDGF) are subject to feedback inhibition via protein kinase C. Images PMID:2519620

  3. Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB/c 3T3 cells.

    PubMed

    Tsuchiya, T; Kato-Masatsuji, E; Tsuzuki, T; Umeda, M

    2000-11-10

    The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB/c 3T3 cells were treated with 0.2 microg/ml 20-methylcholanthrene as an initiator, and 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of L-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; L (+)-ascorbic acid-2-sulfate ester disodium 2H(2)O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor alpha (TNF-alpha) were used as promoters, APM also effectively suppressed transformation. PMID:11098084

  4. Effects of intermedin on proliferation, apoptosis and the expression of OPG/RANKL/M-CSF in the MC3T3-E1 osteoblast cell line

    PubMed Central

    REN, HONGFEI; REN, HONGYU; LI, XUE; YU, DONGDONG; MU, SHUAI; CHEN, ZHIGUANG; FU, QIN

    2015-01-01

    Bone remodeling is a vital physiological process of healthy bone tissue in humans. It is characterized by the formation of bone by osteoblasts and its resorption by osteoclasts, and the bone resorbed by osteoclasts is replaced through the differentiation and activity of osteoblasts. Imbalances in this vital process lead to pathological conditions, including osteoporosis. Intermedin (IMD) as a newly discovered peptide in the calcitonin (CT) family of peptides, which shares similar functions with CT, calcitonin gene-related peptide and amylin in bone resorption. However, the mechanism underlying its effect remains to be elucidated. This was investigated in the present study using the osteoblastic MC3T3-E1 cell line, which was treated with different doses of IMD (0, 1, 10 and 100 nM). Cell proliferation, apoptosis and the expression of receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG) and macrophage colony-stimulating factor (M-CSF) were measured following treatment using multiple detection techniques, including an MTT assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction and western blot analysis. The resulting data demonstrated that IMD significantly inhibited the apoptosis of MC3T3-E1 cells induced by serum-free culture and dexamethasone, however, no significant effects on MC3T3-E1 cell proliferation were observed. IMD had additional functions on the MC3T3-E1 cells, including inhibition of the expression of RANKL and M-CSF, and promotion of the expression of OPG. Previous studies have also demonstrated that RANKL and M-CSF are two vital factor produced by osteoblasts to promote the maturation and differentiation of osteoclasts, and it has been reported that IMD can inhibit the osteoclast formation stimulated by RANKL and M-CSF. Together with these findings, the present study concluded that IMD reduces bone resorption by inhibiting osteoblast apoptosis, decreasing the RANKL/OPG ratio and the expression of M-CSF, and

  5. Traditional Korean Herbal Formula Samsoeum Attenuates Adipogenesis by Regulating the Phosphorylation of ERK1/2 in 3T3-L1 Cells

    PubMed Central

    Jeong, Soo-Jin; Yoo, Sae-Rom; Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Adipogenesis is the cell differentiation process from preadipocytes into adipocytes and the critical action in the development of obesity. In the present study, we conducted in vitro analyses to investigate the inhibitory effects of Samsoeum (SSE), a traditional herbal decoction. SSE had no significant cytotoxic effect against either the undifferentiated or differentiated 3T3-L1 cells. Oil Red O staining results showed that SSE significantly inhibited fat accumulation in adipocytes. SSE treatment consistently reduced the intracellular triglyceride content in the cells. SSE significantly inactivated glycerol-3-phosphate dehydrogenase (GPDH), a major link between carbohydrate and lipid metabolisms in 3T3-L1 adipocytes, and markedly inhibited the production of leptin, an important adipokine, in differentiated cells. SSE markedly suppressed the mRNA expression of the adipogenesis-related genes peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer binding protein-alpha (C/EBP-α), fatty acid synthase (FAS), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). Importantly, SSE increased the phosphorylation of ERK1/2, but not p38 MAPK and JNK, in adipose cells. Overall, our results indicate that SSE exerts antiadipogenic activity and modulates expressions of adipogenesis-related genes and ERK1/2 activation in adipocytes. PMID:26483846

  6. Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling.

    PubMed

    Pengjam, Yutthana; Madhyastha, Harishkumar; Madhyastha, Radha; Yamaguchi, Yuya; Nakajima, Yuichi; Maruyama, Masugi

    2016-03-01

    Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific.Alizarin red S staining revealed a signifiant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influenceof aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influenceof aloin on ALP activity, confirmingthat aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway. PMID:26869456

  7. Anthraquinone Glycoside Aloin Induces Osteogenic Initiation of MC3T3-E1 Cells: Involvement of MAPK Mediated Wnt and Bmp Signaling

    PubMed Central

    Pengjam, Yutthana; Madhyastha, Harishkumar; Madhyastha, Radha; Yamaguchi, Yuya; Nakajima, Yuichi; Maruyama, Masugi

    2016-01-01

    Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway. PMID:26869456

  8. Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

    PubMed Central

    Mei, Gang; Zou, Zhenlv; Fu, Su; Xia, Liheng; Zhou, Jian; Zhang, Yongtao; Tuo, Yonghua; Wang, Zhao; Jin, Dan

    2014-01-01

    Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10−10 to 10−8 M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10−9 to 10−8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10−8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10−8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway. PMID:24733069

  9. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC ; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  10. The left half of the XMRV retrovirus is present in an endogenous retrovirus of NIH/3T3 Swiss mouse cells.

    PubMed

    Mendoza, Ramon; Vaughan, Andrew E; Miller, A Dusty

    2011-09-01

    Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus found in association with human prostate cancer and chronic fatigue syndrome, although these associations are controversial. XMRV shows at most 94% identity to known mouse retroviruses. Here we used XMRV-specific PCR to search for a more closely related source of XMRV in mice. While we could not find a complete copy, we did find a 3,600-bp region of XMRV in an endogenous retrovirus present in NIH/3T3 cells. These results show that XMRV has clear ancestors in mice and highlight another possible source of contamination in PCR assays for XMRV. PMID:21697491

  11. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    PubMed

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  12. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells

    PubMed Central

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2014-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC–MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  13. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approx. 50-fold and their carboxypeptidase. A content approx. 100-fold, and augment approx. their biosynthesis of proteoglycans bearing /sup 35/S-labeled haparin relative to /sup 35/S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

  14. Sheets of Vertically Aligned BaTiO3 Nanotubes Reduce Cell Proliferation but Not Viability of NIH-3T3 Cells

    PubMed Central

    Giannini, Marianna; Giannaccini, Martina; Sibillano, Teresa; Giannini, Cinzia; Liu, Dun; Wang, Zhigang; Baù, Andrea; Dente, Luciana; Cuschieri, Alfred; Raffa, Vittoria

    2014-01-01

    All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants. PMID:25506693

  15. N(ϵ) -Carboxymethyllysine Increases the Expression of miR-103/143 and Enhances Lipid Accumulation in 3T3-L1 Cells.

    PubMed

    Holik, Ann-Katrin; Lieder, Barbara; Kretschy, Nicole; Somoza, Mark M; Held, Sandra; Somoza, Veronika

    2016-10-01

    Advanced glycation endproducts, formed in vivo, but also by the Maillard reaction upon thermal treatment of foods, have been associated with the progression of pathological conditions such as diabetes mellitus. In addition to the accumulation with age, exogenous AGEs are introduced into the circulation from dietary sources. In this study, we investigated the effects of addition of free N(ϵ) -carboxymethyllysine (CML), a well-characterized product of the Maillard reaction, on adipogenesis in 3T3-L1 preadipocytes. Treatment with 5, 50, or 500 μM CML resulted in increased lipid accumulation to similar extents, by 11.5 ± 12.6%, 12.9 ± 8.6%, and 12.8 ± 8.5%, respectively. Long-term treatment with 500 μM CML during adipogenesis resulted in increases in miR-103 and miR-143 levels, two miRNAs described to be involved in impaired glucose homeostasis and increased lipid accumulation. Furthermore, the expression of genes associated with these miRNAs, consisting of Akt1, PI3k, and Cav1 was regulated by CML. Short-term treatment of mature 3T3-L1 adipocytes with CML resulted in decreased basal glucose uptake. These results, indicate that the addition of protein-free CML to 3T3-L1 cells influence parameters associated with adipogenesis and glucose homeostasis at transcriptional, and functional level; this indicates that free CML derived from exogenous sources, in addition to protein-bound CML may be relevant in this context. J. Cell. Biochem. 117: 2413-2422, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27137869

  16. Nϵ‐Carboxymethyllysine Increases the Expression of miR‐103/143 and Enhances Lipid Accumulation in 3T3‐L1 Cells

    PubMed Central

    Holik, Ann‐Katrin; Lieder, Barbara; Kretschy, Nicole; Somoza, Mark M.; Held, Sandra

    2016-01-01

    ABSTRACT Advanced glycation endproducts, formed in vivo, but also by the Maillard reaction upon thermal treatment of foods, have been associated with the progression of pathological conditions such as diabetes mellitus. In addition to the accumulation with age, exogenous AGEs are introduced into the circulation from dietary sources. In this study, we investigated the effects of addition of free Nϵ‐carboxymethyllysine (CML), a well‐characterized product of the Maillard reaction, on adipogenesis in 3T3‐L1 preadipocytes. Treatment with 5, 50, or 500 μM CML resulted in increased lipid accumulation to similar extents, by 11.5 ± 12.6%, 12.9 ± 8.6%, and 12.8 ± 8.5%, respectively. Long‐term treatment with 500 μM CML during adipogenesis resulted in increases in miR‐103 and miR‐143 levels, two miRNAs described to be involved in impaired glucose homeostasis and increased lipid accumulation. Furthermore, the expression of genes associated with these miRNAs, consisting of Akt1, PI3k, and Cav1 was regulated by CML. Short‐term treatment of mature 3T3‐L1 adipocytes with CML resulted in decreased basal glucose uptake. These results, indicate that the addition of protein‐free CML to 3T3‐L1 cells influence parameters associated with adipogenesis and glucose homeostasis at transcriptional, and functional level; this indicates that free CML derived from exogenous sources, in addition to protein‐bound CML may be relevant in this context. J. Cell. Biochem. 117: 2413–2422, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:27137869

  17. Antioxidant action and cytotoxicity on HeLa and NIH-3T3 cells of new quercetin derivatives

    PubMed Central

    Veverka, Miroslav; Šturdík, Ernest; Jantová, Soňa

    2013-01-01

    Quercetin is a natural polyphenol with proven health beneficial activities. In this study 15 new quercetin derivatives were prepared with the aim to enhance their bioavailability. Modification of their physicochemical properties could herewith improve the action in cells. The prepared compounds were tested for their antioxidant and cytotoxic activity. The ability to scavenge free radicals as well as ferric reducing antioxidant power of the new derivatives was not better than that of unmodified quercetin. But for acetylated esters a better cytotoxic activity was found on human cervical cancer cells HeLa than for the initial molecule. The best effect revealed chloronaphtoquinone quercetin (IC50=13.2 µM). For this compound comparable cytotoxic action on non-cancer murine fibroblast cells was detected (IC50=16.5 µM). The obtained results indicate that appropriate lipophilization of the quercetin molecule could improve its cytotoxic action in cells, probably due to its enhanced bioavailability. PMID:24678260

  18. Poly(acrylic acid)-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells.

    PubMed

    Yang, Wei; Yao, Chenxue; Cui, Zhengyang; Luo, Dandan; Lee, In-Seop; Yao, Juming; Chen, Cen; Kong, Xiangdong

    2016-01-01

    Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of -22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration. PMID:27164090

  19. Green Tea (-)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes.

    PubMed

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-03-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The -970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  20. Green Tea (−)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes

    PubMed Central

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-01-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The −970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  1. Poly(acrylic acid)-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells

    PubMed Central

    Yang, Wei; Yao, Chenxue; Cui, Zhengyang; Luo, Dandan; Lee, In-Seop; Yao, Juming; Chen, Cen; Kong, Xiangdong

    2016-01-01

    Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs) with desired water dispersibility were achieved with the regulation of poly (acrylic acid). Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of −22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS) solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP) activity assays together with the osteocalcin (OCN) and bone sialoprotein (BSP) expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration. PMID:27164090

  2. Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

    PubMed

    Fatokun, Amos A; Tome, Mercedes; Smith, Robert A; Darlington, L Gail; Stone, Trevor W

    2015-01-01

    Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway. PMID:25427161

  3. Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell in vitro transformation assay for eight phthalate esters.

    PubMed

    Barber, E D; Cifone, M; Rundell, J; Przygoda, R; Astill, B D; Moran, E; Mulholland, A; Robinson, E; Schneider, B

    2000-01-01

    Eight phthalate esters, with alcohol chain lengths of 1-11 carbon atoms and with various degrees of branching, were tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay and in the Balb/3T3 cell transformation assay. The tests were performed as part of a voluntary testing agreement between the Chemical Manufacturers Association's Phthalate Esters Panel and the United States Environmental Protection Agency (US EPA). The esters tested were: dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di-¿n-hexyl, n-octyl, n-decyl¿ phthalate (610P), di-isononyl phthalate (DINP), di-¿heptyl, nonyl, undecyl¿ phthalate (711P), di-isodecyl phthalate (DIDP) and di-undecyl phthalate (DUP). Both DMP and DBP were found to produce significant increases in the mutant frequency in the mouse lymphoma assay in the presence but not in the absence of an Aroclor-induced rat liver activation system (S-9). Ester 610P gave equivocal results in the mouse lymphoma assay in the presence and absence of rat liver S-9. There was no indication of mutagenic potential for any of the other test materials in the mouse lymphoma assay, and none of the test materials increased transformation frequency in the Balb/3T3 cell transformation assay. Aldehyde metabolites of the de-esterified alcohols are postulated to play a role in the positive results for DMP and DBP. PMID:10641018

  4. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    NASA Astrophysics Data System (ADS)

    Li, Huihua; Luo, Chuang; Luo, Binghong; Wen, Wei; Wang, Xiaoying; Ding, Shan; Zhou, Changren

    2016-01-01

    To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  5. Production of bioactive enkephalin from the nonendocrine cell lines COS-7, NIH3T3, Ltk-, and C2C12.

    PubMed

    Takahashi, K; Fujita, T; Takeuchi, T

    1995-01-01

    Enkephalin is synthesized from proenkephalin in neuroendocrine cells. For the attempt to induce nonneuroendocrine origin cells to produce enkephalin, we used a mammalian expression vector for fusion peptides, pMEproCT beta, in which a fused peptide is designed to be cleaved by a yeast Kex2-like endoprotease furin. Met-Enkephalin was expressed in four nonneuroendocrine cell lines: COS-7, C2C12, Ltk-, and NIH3T3. The four cell lines produced a marked amount of Met-enkephalin, which appeared as a single peak on reverse-phase HPLC. Because transplantation of adrenal medullary cells to the subarachnoid space has been used to alleviate terminal cancer pain, and enkephalin appears to play a central role in relieving pain, this enkephalin expression vector may be useful for direct enkephalin expression in pericancerous tissues. PMID:7479338

  6. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  7. Arctiin inhibits adipogenesis in 3T3-L1 cells and decreases adiposity and body weight in mice fed a high-fat diet

    PubMed Central

    Min, Byulchorong; Lee, Heejin; Song, Ji Hye; Han, Myung Joo

    2014-01-01

    BACKGROUND/OBJECTIVES The purpose of this study was to examine the effects and associated mechanisms of arctiin, a lignan compound found in burdock, on adipogenesis in 3T3-L1 cells. Also, the effects of arctiin supplementation in obese mice fed a high-fat diet on adiposity were examined. MATERIALS/METHODS 3T3-L1 cells were treated with arctiin (12.5 to 100 µM) during differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining and intracellular triglyceride contents. The expressions of genes related to adipogenesis were measured by real-time RT-PCR and Western blot analyses. For in vivo study, C57BL/6J mice were first fed either a control diet (CON) or high-fat diet (HF) to induce obesity, and then fed CON, HF, or HF with 500 mg/kg BW arctiin (HF + AC) for four weeks. RESULTS Arctiin treatment to 3T3-L1 pre-adipocytes markedly decreased adipogenesis in a dose-dependent manner. The arctiin treatment significantly decreased the protein levels of the key adipogenic regulators PPARγ and C/EBPα, and also significantly inhibited the expression of SREBP-1c, fatty acid synthase, fatty acid-binding protein and lipoprotein lipase. Also, arctiin greatly increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase. Furthermore, administration of arctiin significantly decreased the body weight in obese mice fed with the high-fat diet. The epididymal, perirenal or total visceral adipose tissue weights of mice were all significantly lower in the HF + AC than in the HF. Arctiin administration also decreased the sizes of lipid droplets in the epididymal adipose tissue. CONCLUSIONS Arctiin inhibited adipogenesis in 3T3-L1 adipocytes through the inhibition of PPARγ and C/EBPα and the activation of AMPK signaling pathways. These findings suggest that arctiin has a potential benefit in preventing obesity. PMID:25489405

  8. Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Cariveau, Mickael J.

    2005-07-01

    Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

  9. Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

    SciTech Connect

    Li, Qiang; Kamemura, Kazuo

    2014-07-18

    Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.

  10. High-Speed Microdialysis-Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells.

    PubMed

    Harstad, Rachel K; Bowser, Michael T

    2016-08-16

    We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay. PMID:27398773

  11. Ca/sup 2 +/-mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells

    SciTech Connect

    Lopez-Rivas, A.; Mendoza, S.A.; Nanberg, E.; Sinnett-Smith, J.; Rozengurt, E.

    1987-08-01

    Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca/sup 2 +/ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca/sup 2 +/ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca/sup 2 +/ movements were also substantiated by measurements of /sup 45/Ca/sup 2 +/ efflux and of cellular /sup 45/Ca/sup 2 +/ content. Activation of protein kinase C by phorbol esters inhibited Ca/sup 2 +/ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca/sup 2 +/ concentration increase or acceleration of /sup 45/Ca/sup 2 +/ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathway activated by PDGF that lead to Ca/sup 2 +/ mobilization can be distinguished form those utilized by bombesin and vasopressin.

  12. Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.

    PubMed

    Ponti, Jessica; Broggi, Francesca; Mariani, Valentina; De Marzi, Laura; Colognato, Renato; Marmorato, Patrick; Gioria, Sabrina; Gilliland, Douglas; Pascual Garcìa, César; Meschini, Stefania; Stringaro, Annarita; Molinari, Agnese; Rauscher, Hubert; Rossi, François

    2013-03-01

    In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity. PMID:22279961

  13. Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebrafish.

    PubMed

    Lee, Yeon-Joo; Choi, Hyeon-Son; Seo, Min-Jung; Jeon, Hui-Jeon; Kim, Kui-Jin; Lee, Boo-Yong

    2015-08-01

    Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression. Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism. PMID:26174858

  14. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    PubMed

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. PMID:3543375

  15. Cytotoxicity of Agaricus sylvaticus in non-tumor cells (NIH/3T3) and tumor (OSCC-3) using tetrazolium (MTT) assay.

    PubMed

    Orsine, Joice Vinhal Costa; Marques Brito, Luíssa; Silva, Renata Carvalho; Santos Almeida, Maria de Fátima Menezes; Novaes, Maria Rita Carvalho Garbi

    2013-01-01

    The purpose of this study was to assess the cytotoxic effect of the non-fractionated aqueous extract of A. sylvaticus mushroom in cultures of non-tumor cells (NIH3T3) and tumor cells (OSCC-3). The cells were maintained in DMEN cell culture medium added of 10% of fetal bovine serum and 1% antibiotic. For the cytotoxicity test we prepared the aqueous mushroom extract at concentrations of 0.01 mg.ml⁻¹, 0.02 mg.ml⁻¹, 0.04 mg.ml⁻¹, 0.08 mg.ml⁻¹, 0.16 mg.ml⁻¹, and 0.32 mg.ml⁻¹. For the culture, 2 x 10⁵ cells/ml was deposited in 96-well microplates during 24 hour incubation with subsequent exchange of medium by another containing the mushroom concentrations. After 24 hour incubation the medium was discarded and 100 ml of tetrazolium blue (MTT) was added at a concentration of 5 mg.ml⁻¹. The microplates were incubated for 2 h at 37° C. Spectrophotometric analysis was performed using 570 nm wavelength. From the values of the optical densities we determined the drug concentration capable of reducing cell viability by 50%. Therefore, the mushroom A. sylvaticus, at all concentrations tested, did not show cytotoxic effects, once the inhibitory concentration (IC₅₀) obtained for tumor cells OSCC-3 was 0.06194 mg.ml⁻¹, and the IC₅₀ checked for non-tumor cells NIH3T3 was 0,06468 mg.ml⁻¹. This test made it possible to determine that A. sylvaticus mushroom has no cytotoxic effects, suggesting its use safe for human consumption. PMID:23889648

  16. CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells.

    PubMed

    Santangelo, Carmela; Varì, Rosaria; Scazzocchio, Beatrice; Filesi, Carmelina; D'Archivio, Massimo; Giovannini, Claudio; Masella, Roberta

    2011-09-01

    Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the

  17. Toddaculin, Isolated from of Toddalia asiatica (L.) Lam., Inhibited Osteoclastogenesis in RAW 264 Cells and Enhanced Osteoblastogenesis in MC3T3-E1 Cells

    PubMed Central

    Watanabe, Akio; Kumagai, Momochika; Mishima, Takashi; Ito, Junya; Otoki, Yurika; Harada, Teppei; Kato, Tsuyoshi; Yoshida, Mikihiko; Suzuki, Misora; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2015-01-01

    Osteoporosis with bone loss is widely recognized as a major health problem. Bone homeostasis is maintained by balancing bone formation and bone resorption. The imbalance caused by increased bone resorption over bone formation can lead to various bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoclasts are the principal cells responsible for bone resorption and the main targets of anti-resorptive therapies. However, excessive inhibition of osteoclast differentiation may lead to inhibition of osteoblast differentiation. Therefore, it is important to screen for new compounds capable of inhibiting bone resorption and enhancing bone formation. Toddalia asiatica (L.) Lam. has been utilized traditionally for medicinal purposes such as the treatment of rheumatism. Currently, the extract is considered to be a good source of pharmacological agents for the treatment of bone-related diseases, but the active compounds have yet to be identified. We investigated whether toddaculin, derived from Toddalia asiatica (L.) Lam., affects both processes by inhibiting bone resorption and enhancing bone formation. Towards this end, we used pre-osteoclastic RAW 264 cells and pre-osteoblastic MC3T3-E1 cells. We found that toddaculin not only inhibited the differentiation of osteoclasts via activation of the NF-κB, ERK 1/2, and p38 MAPK signaling pathways, but it also induced differentiation and mineralization of osteoblasts by regulating differentiation factors. Thus, toddaculin might be beneficial for the prevention and treatment of osteoporosis. PMID:25993011

  18. Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures.

    PubMed

    Fu, Qiang; Rahaman, Mohamed N; Bal, B Sonny; Brown, Roger F

    2009-05-01

    Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique 'elastic-plastic' mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65-70%) but different pore architectures, described as 'lamellar' (pore width = 25 +/- 5 microm) and 'cellular' (pore diameter = 100 +/- 10 microm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications. PMID:19115092

  19. miR-103 promotes 3T3-L1 cell adipogenesis through AKT/mTOR signal pathway with its target being MEF2D.

    PubMed

    Li, Meihang; Liu, Zhenjiang; Zhang, Zhenzhen; Liu, Guannv; Sun, Shiduo; Sun, Chao

    2015-03-01

    MicroRNAs are small non-coding RNAs that partially bind to the 3' untranslated (3'UTR) regions of target genes in animals and regulate protein production of the target transcripts. MiR-103 has been confirmed to play a critical role in lipid metabolism, however, the target genes and signaling pathway regulated by miR-103 is still unclear. In our experiment, we observed a positive function of miR-103 on the adipogenic differentiation of 3T3-L1 pre-adipocyte. Furthermore, we proved that this function of miR-103 worked through activating AKT/mTOR signal pathway and impairing target gene MEF2D. By inhibiting and over-expressing the MEF2D gene, we found that MEF2D had a negative role in regulating adipocyte key genes, and this function of MEF2D could be impaired by miR-103. In conclusion, we found that miR-103 can promote 3T3-L1 cells differentiation by targeting MEF2D and activating AKT/mTOR signal pathway. These results will shed a light on further study of microRNAs. PMID:25400071

  20. Adipogenic effects of piperlonguminine in 3T3-L1 cells and plasma concentrations of several amide constituents from Piper chaba extracts after treatment of mice.

    PubMed

    Yamaguchi, Itadaki; Matsuda, Hisashi; Zhang, Hailong; Hamao, Makoto; Yamashita, Chihiro; Kogami, Yuichiro; Kon'I, Haruka; Murata, Megumi; Nakamura, Seikou; Yoshikawa, Masayuki

    2014-01-01

    In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-γ (PPARγ) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPARγ2 but, unlike troglitazone, piperlonguminine did not activate PPARγ directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract. PMID:23584920

  1. New pyrano-pyrone from Goniothalamus tamirensis enhances the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

    PubMed

    Tai, Bui Huu; Huyen, Vu Thi; Huong, Tran Thu; Nhiem, Nguyen Xuan; Choi, Eun-Mi; Kim, Jeong Ah; Long, Pham Quoc; Cuong, Nguyen Manh; Kim, Young Ho

    2010-04-01

    The new pyrano-pyrone, (+)-8-epi-9-deoxygoniopypyrone (1) and (+)-9-deoxygoniopypyrone (2) were isolated from a chloroform extract of Goniothalamus tamirensis leaves. Their absolute stereostructures were discussed and confirmed by using infrared (IR), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), one (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectra, Mosher's method, and comparison with the known compounds leiocapin A (3), deoxygoniopypyrone A (4), and (-)-8-epi-9-deoxygoniopypyrone (5). At concentrations of 2.67 microM, compounds 1 and 2 significantly increased the growth of osteoblastic MC3T3-E1 cells and caused a significant elevation of collagen content, alkaline phosphatase activity, and nodule mineralization in the cells (p<0.05). Our data suggest that the enhancement of osteoblast function by 1 and 2 may result in the prevention of osteoporosis. PMID:20410636

  2. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C

    SciTech Connect

    Zurier, B.B.; Kozma, M.; Sinnett-Smith, J.; Rozengurt, E. )

    1988-05-01

    Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.

  3. Substance P antagonist also inhibits specific binding and mitogenic effects of vasopressin and bombesin-related peptides in Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Rozengurt, E.

    1986-05-29

    While vasopressin and peptides of the bombesin family bind to different receptors in quiescent Swiss 3T3 cells, the antagonist (D-Arg/sup 1/,D-Pro/sup 2/,D-Trp/sup 7,9/,Leu/sup 11/) substance P blocks the specific binding of both (/sup 3/H) vasopressin and /sup 125/I-gastrin-releasing peptide to these cells. In addition, the antagonist inhibits the mobilization of Ca/sup 2 +/ and induction of DNA synthesis by vasopressin. These results indicate that (D-Arg/sup 1/,D-Pro,D-Trp/sup 7,9/,Leu/sup 11/) substance P has the ability to interact with the receptors for three structurally unrelated peptide hormones.

  4. Isolation of up- or down-regulated genes in PPARgamma-expressing NIH-3T3 cells during differentiation into adipocytes.

    PubMed

    Okuno, Masaaki; Arimoto, Emi; Nishizuka, Makoto; Nishihara, Tsutomu; Imagawa, Masayoshi

    2002-05-22

    Adipocyte differentiation is a complex process in which the expression of many transcription factors and adipocyte-specific genes is regulated under a strict program. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear hormone receptor superfamily of ligand-activated transcription factors, is an important regulator of adipocyte gene expression and differentiation. In this study, we tried to identify downstream target genes of PPARgamma, by using PPARgamma-expressing cells and a subtractive cloning strategy, and isolated cDNA clones which were up-regulated or down-regulated by PPARgamma. Northern blot analyses revealed that the expression levels of the aldehyde dehydrogenase-2-like, type VI collagen alpha 3 subunit, cellular retinoic acid binding protein 1 and thrombospondin 1 are changed during the differentiation of mouse 3T3-L1 preadipocyte cells, indicating that these genes might be downstream targets of PPARgamma in adipocyte differentiation. PMID:12023027

  5. The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces.

    PubMed

    Lumetti, S; Manfredi, E; Ferraris, S; Spriano, S; Passeri, G; Ghiacci, G; Macaluso, G; Galli, C

    2016-04-01

    The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation. PMID:26886816

  6. Inhibitory effects of constituents from Euphorbia lunulata on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells.

    PubMed

    Yang, Zhi-Gang; Jia, Liu-Nan; Shen, Yan; Ohmura, Atsuko; Kitanaka, Susumu

    2011-01-01

    A new flavonol galactopyranoside, myricetin 3-O-(2'',3''-digalloyl)-β-D-galactopyranoide (1), and 23 known constituents, including myricetin 3-O-(2''-galloyl)-β-D-galactopyranoide (2), myricitrin (3), myricetin (4), quercetin 3-O-(2'', 3''-digalloyl)-β-D-galactopyranoide (5), quercetin 3-O-(2''-galloyl)-β-D-galactopyranoide (6), hyperin (7), isoquercetrin (8), quercetin (9), kaempferol (10), apigenin (11), luteolin (12), 3-O-methylquercetin (13), 5,7,2',5'-tetrahydroxyflavone (14), 1,3,4,6-tetra-O-galloyl-β-D-glucose (15), 1,2,6-tri-O-galloyl-β-D-glucose (16), 1,3,6-tri-O-galloyl-β-D-glucose (17), gallic acid (18), protocatechuic acid (19), 3,4,5-trimethoxybenzoic acid (20), 2,6-dihydroxyacetophenone (21), 3,3'-di-O-methylellagic acid (22), ellagic acid (23) and esculetin (24) were isolated from Euphorbia lunulata Bge. Their structures were determined by spectroscopic analysis. Isolated hydrolysable tannins, flavonoids, and flavonol galactopyranoside gallates showed significant inhibition of the differentiation of 3T3-L1 preadipocytes and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW 264.7 cells. PMID:21959301

  7. Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells.

    PubMed

    Yang, Zhi-Gang; Matsuzaki, Keiichi; Takamatsu, Satoshi; Kitanaka, Susumu

    2011-01-01

    A new arylbenzofuran, 3',5'-dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran (1), and 25 known compounds, including moracin R (2), moracin C (3), moracin O (4), moracin P (5), artoindonesianin O (6), moracin D (7), alabafuran A (8), mulberrofuran L (9), mulberrofuran Y (10), kuwanon A (11), kuwanon C (12), kuwanon T (13), morusin (14), kuwanon E (15), sanggenon F (16), betulinic acid (17), uvaol (18), ursolic acid (19), β-sitosterol (20), oxyresveratrol 2-O-β-D-glucopyranoside (21), mulberroside A (22), mulberroside B (23), 5,7-dihydroxycoumarin 7-O-β-D-glucopyranoside (24), 5,7-dihydroxycoumarin 7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (25) and adenosine (26), were isolated from Morus alba var. multicaulis Perro. (Moraceae). Their structures were determined by spectroscopic methods. The prenyl-flavonoids 11-14, 16, triterpenoids 17,18 and 20 showed significant inhibitory activity towards the differentiation of 3T3-L1 adipocytes. The arylbenzofurans 1-10 and prenyl-flavonoids 11-16 also showed significant nitric oxide (NO) production inhibitory effects in RAW264.7 cells. PMID:21772233

  8. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

    PubMed

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-03-16

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  9. Bezafibrate prevents palmitate-induced apoptosis in osteoblastic MC3T3-E1 cells through the NF-κB signaling pathway.

    PubMed

    Zhong, Xing; Xiu, Lingling; Wei, Guohong; Pan, Tianrong; Liu, Yuanyuan; Su, Lei; Li, Yanbing; Xiao, Haipeng

    2011-10-01

    Osteoporosis is a bone condition defined by low bone mass and increase of fracture risk due to imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Low bone mass is likely to be due to the alteration of the osteoclast and osteoblast lifespan through regulated apoptosis. Saturated fatty acid (SFA) intake is negatively associated with bone mineral density (BMD). Furthermore, SFA induces apoptosis in osteoblastic cell lines. Bezafibrate could increase bone mass in intact male rats principally through increasing periosteal bone formation. At present, it is unknown whether bezafibrate attenuates palmitate-induced apoptosis in MC3T3-E1 cells. In the present study, we found that palmitate stimulated the degradation of IκBα and NF-κB translocation, as well as up-regulation of NF-κB-mediated Fas expression in obsteoblastic MC3T3-E1 cells. Furthermore, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could restore palmitate-induced caspase-3 decrease and inhibit palmitate-induced cleaved caspase-3 increase. We observed that bezafibrate, a dual ligand for the peroxisome proliferator-activated receptors α (PPARα) and PPARδ, significantly attenuated the palmitate-induced cytotoxicity as determined by the MTT assay and inhibited the palmitate-induced apoptosis as determined by a flow cytometry assay using Annexin V-FITC/PI and assessment of the activity of caspase-3. Pre-treatment of bezafibrate prevented palmitate-induced NF-κB activation. Therefore, these findings indicate that bezafibrate inbibits palmitate-induced apoptosis via the NF-κB signaling pathway. Our results point to bezafibrate as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions. PMID:21687928

  10. Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts*

    PubMed Central

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-01-01

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  11. Inhibition of acetyl-CoA carboxylases by soraphen A prevents lipid accumulation and adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Cordonier, Elizabeth L; Jarecke, Sarah K; Hollinger, Frances E; Zempleni, Janos

    2016-06-01

    Acetyl-CoA carboxylases (ACC) 1 and 2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA and depend on biotin as a coenzyme. ACC1 localizes in the cytoplasm and produces malonyl-CoA for fatty acid (FA) synthesis. ACC2 localizes in the outer mitochondrial membrane and produces malonyl-CoA that inhibits FA import into mitochondria for subsequent oxidation. We hypothesized that ACCs are checkpoints in adipocyte differentiation and tested this hypothesis using the ACC1 and ACC2 inhibitor soraphen A (SA) in murine 3T3-L1 preadipocytes. When 3T3-L1 cells were treated with 100nM SA for 8 days after induction of differentiation, the expression of PPARγ mRNA and FABP4 mRNA decreased by 40% and 50%, respectively, compared with solvent controls; the decrease in gene expression was accompanied by a decrease in FABP4 protein expression and associated with a decrease in lipid droplet accumulation. The rate of FA oxidation was 300% greater in SA-treated cells compared with vehicle controls. Treatment with exogenous palmitate restored PPARγ and FABP4 mRNA expression and FABP4 protein expression in SA-treated cells. In contrast, SA did not alter lipid accumulation if treatment was initiated on day eight after induction of differentiation. We conclude that loss of ACC1-dependent FA synthesis and loss of ACC2-dependent inhibition of FA oxidation prevent lipid accumulation in adipocytes and inhibit early stages of adipocyte differentiation. PMID:27041646

  12. Ionomycin induces prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells via mechanisms independent of its ionophoric nature.

    PubMed

    Leis, Hans Jörg; Windischhofer, Werner

    2016-06-01

    Ionomycin and A23187 are divalent cation ionophores with a marked preference for calcium. Studies using these ionophores have almost exclusively interpreted their results in the light of calcium elevation. It was the aim of this study to investigate the effects of ionomycin in osteoblatic MC3T3-E1 cells that are not attributable to its ionophoric properties. Thus, we have found that in contrast to A23187, ionomycin shows similar effects on prostaglandin E2 formation as bradykinin and endothelin-1, being potentiated by extracellular nickel and inhibited by cholera toxin and pertussis toxin. Our data strongly suggest that inomycin, at least in part, exerts its effects via specific binding to a G-protein coupled receptor, thereby evoking downstream cellular events like arachidonate release with subsequent prostaglandin formation. PMID:27065246

  13. ToF-SIMS depth profiling of cells: z-correction, 3D imaging, and sputter rate of individual NIH/3T3 fibroblasts.

    PubMed

    Robinson, Michael A; Graham, Daniel J; Castner, David G

    2012-06-01

    Proper display of three-dimensional time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging data of complex, nonflat samples requires a correction of the data in the z-direction. Inaccuracies in displaying three-dimensional ToF-SIMS data arise from projecting data from a nonflat surface onto a 2D image plane, as well as possible variations in the sputter rate of the sample being probed. The current study builds on previous studies by creating software written in Matlab, the ZCorrectorGUI (available at http://mvsa.nb.uw.edu/), to apply the z-correction to entire 3D data sets. Three-dimensional image data sets were acquired from NIH/3T3 fibroblasts by collecting ToF-SIMS images, using a dual beam approach (25 keV Bi(3)(+) for analysis cycles and 20 keV C(60)(2+) for sputter cycles). The entire data cube was then corrected by using the new ZCorrectorGUI software, producing accurate chemical information from single cells in 3D. For the first time, a three-dimensional corrected view of a lipid-rich subcellular region, possibly the nuclear membrane, is presented. Additionally, the key assumption of a constant sputter rate throughout the data acquisition was tested by using ToF-SIMS and atomic force microscopy (AFM) analysis of the same cells. For the dried NIH/3T3 fibroblasts examined in this study, the sputter rate was found to not change appreciably in x, y, or z, and the cellular material was sputtered at a rate of approximately 10 nm per 1.25 × 10(13) ions C(60)(2+)/cm(2). PMID:22530745

  14. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

  15. Effects of uremic toxin p-cresol on proliferation, apoptosis, differentiation, and glucose uptake in 3T3-L1 cells.

    PubMed

    Tanaka, Sayuri; Yano, Shozo; Sheikh, Abdullah M; Nagai, Atsushi; Sugimoto, Toshitsugu

    2014-07-01

    Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500 μM 3-isobutyl-methylxanthine, 250 nM dexamethasone, and 10 μg/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200 μM. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200 μM p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200 μM p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200 μM p-cresol. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased

  16. Cell-free Embryonic Stem Cell Extract-Mediated Derivation of Multi-potent Stem Cells from NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

    PubMed Central

    Rajasingh, Johnson; Lambers, Erin; Hamada, Hiromichi; Bord, Evelyn; Thorne, Tina; Goukassian, Ilona; Krishnamurthy, Prasanna; Rosen, Kenneth M.; Ahluwalia, Deepali; Zhu, Yan; Qin, Gangjian; Losordo, Douglas W.; Kishore, Raj

    2008-01-01

    The oocyte-independent source for the generation of pluripotent stem cells is one of the ultimate goals in regenerative medicine. We report that upon exposure to mouse ES cell (ESC) extracts, reversibly permeabilized NIH3T3 cells undergo de-differentiation followed by stimulus-induced re-differentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 control and ESC extract treated NIH3T3 cells including the re-activation of ESC specific transcripts. Epigenetically, ESC extracts induced CpG de-methylation of Oct4 promoter, hyper-acetylation of histones 3 and 4 and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically-induced hind limb ischemia (HLI) or acute myocardial infarction (AMI) transplantation of reprogrammed NIH3T3 cells significantly improved post-injury physiological functions and showed anatomical evidence of engraftment and trans-differentiation into skeletal muscle, endothelial cell and cardiomyocytes. These data provide evidence for the generation of functional multi-potent stem like cells from terminally differentiated somatic cells without the introduction of retroviral mediated trans-genes or ESC fusion. PMID:18483406

  17. Cells (MC3T3-E1)-laden alginate scaffolds fabricated by a modified solid-freeform fabrication process supplemented with an aerosol spraying.

    PubMed

    Ahn, SeungHyun; Lee, HyeongJin; Bonassar, Lawrence J; Kim, GeunHyung

    2012-09-10

    In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl(2)), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 μm) and an aerosol spray flow rate (0.93 ± 0.12 mL min(-1)), 10 mm s(-1) nozzle moving speed, a 10 wt % concentration of CaCl(2) in the aerosol solution, and 2 wt % concentration of CaCl(2) in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, pore-structured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm(3) and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well. PMID:22913233

  18. Alpha-Lipoic Acid Promotes Osteoblastic Formation in H2O2 -Treated MC3T3-E1 Cells and Prevents Bone Loss in Ovariectomized Rats.

    PubMed

    Fu, Chao; Xu, Dong; Wang, Chang-Yuan; Jin, Yue; Liu, Qi; Meng, Qiang; Liu, Ke-Xin; Sun, Hui-Jun; Liu, Mo-Zhen

    2015-09-01

    Alpha-lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. Recently, accumulating evidences has indicated the relationship between oxidative stress and osteoporosis (OP). Some studies have investigated the possible beneficial effects of ALA on OP both in vivo and in vitro; however, the precise mechanism(s) underlying the bone-protective action of ALA remains unclear. Considering this, we focused on the anti-oxidative capacity of ALA to exert bone-protective effects in vitro and in vivo. In the present study, the effects of ALA on osteoblastic formation in H(2)O(2) -treated MC3T3-E1 pre-osteoblasts and ovariectomy (OVX)-induced bone loss in rats were investigated. The results showed that ALA promoted osteoblast differentiation, mineralization and maturation and inhibited osteoblast apoptosis, thus increasing the OPG/receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) ratio and leading to enhanced bone formation in vitro and inhibited bone loss in vivo. Further study revealed that ALA exerted its bone-protective effects by inhibiting reactive oxygen species (ROS) generation by down-regulating Nox4 gene expression and protein synthesis and attenuating the transcriptional activation of NF-κB. In addition, ALA might exert its bone-protective effects by activating the Wnt/Lrp5/β-catenin signaling pathway. Taken together, the present study indicated that ALA promoted osteoblastic formation in H(2)O(2) -treated MC3T3-E1 cells and prevented OVX-induced bone loss in rats by regulating Nox4/ROS/NF-κB and Wnt/Lrp5/β-catenin signaling pathways, which provided possible mechanisms of bone-protective effects in regulating osteoblastic formation and preventing bone loss. Taken together, the results suggest that ALA may be a candidate for clinical OP treatment. PMID:25655087

  19. Assessment of cytocompatibility of surface-modified CdSe/ZnSe quantum dots for BALB/3T3 fibroblast cells.

    PubMed

    Mahto, Sanjeev Kumar; Park, Chansik; Yoon, Tae Hyun; Rhee, Seog Woo

    2010-06-01

    With the widespread use of quantum dots (QDs), the likelihood of exposure to QDs has been assumed to have increased substantially. Recently, QDs have been employed in numerous biological and medical applications. However, there is a lack of toxicological data pertaining to QDs. In this study, we aimed to investigate the cytocompatibility of surface-modified CdSe/ZnSe QDs for BALB/3T3 fibroblast cells. The ligands used for surface modification are mercaptopropionic acid (MPA) and Gum arabic (GA)/tri-n-octylphosphine oxide (TOPO). Cells were exposed to different concentrations of QDs followed by illustrative cytotoxicity analyses. Furthermore, we used a confocal microscope to assess intracellular uptake of QDs. Confocal images showed that MPA-coated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPO-coated QDs were not found inside cells. MPA-coated QDs were highly cytocompatible, whereas GA/TOPO-coated QDs were toxic to the cells. Cells treated with GA/TOPO-coated QDs showed altered morphology, decreased viability, significant concentrations of intracellular free cadmium, detectable reactive oxygen species (ROS) formation, depolymerized cytoskeleton, and irregular-shaped nuclei. This study suggests that surface modification by ligands plays a significant role in the prevention of cytotoxicity of QDs. PMID:20362659

  20. Vitronectin absorbed on nanoparticles mediate cell viability/proliferation and uptake by 3T3 Swiss albino mouse fibroblasts: in vitro study.

    PubMed

    Rosso, F; Marino, G; Grimaldi, A; Cafiero, G; Chiellini, E; Chiellini, F; Barbarisi, M; Barbarisi, A

    2013-01-01

    We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 μg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation. PMID:23710450

  1. Exposure to airborne PM2.5 suppresses microRNA expression and deregulates target oncogenes that cause neoplastic transformation in NIH3T3 cells

    PubMed Central

    Cheng, Xinxin; Shao, Mingming; Wu, Chen; Wang, Suhan; Li, Hongmin; Wei, Lixuan; Gao, Yanning; Tan, Wen; Cheng, Shujun; Wu, Tangchun; Yu, Dianke; Lin, Dongxin

    2015-01-01

    Long-term exposure to airborne PM2.5 is associated with increased lung cancer risk but the underlying mechanism remains unclear. We characterized global microRNA and mRNA expression in human bronchial epithelial cells exposed to PM2.5 organic extract and integrally analyzed microRNA-mRNA interactions. Foci formation and xenograft tumorigenesis in mice with NIH3T3 cells expressing genes targeted by microRNAs were performed to explore the oncogenic potential of these genes. We also detected plasma levels of candidate microRNAs in subjects exposed to different levels of air PM2.5 and examined the aberrant expression of genes targeted by these microRNAs in human lung cancer. Under our experimental conditions, treatment of cells with PM2.5 extract resulted in downregulation of 138 microRNAs and aberrant expression of 13 mRNAs (11 upregulation and 2 downregulation). In silico and biochemical analyses suggested SLC30A1, SERPINB2 and AKR1C1, among the upregulated genes, as target for miR-182 and miR-185, respectively. Ectopic expression of each of these genes significantly enhanced foci formation in NIH3T3 cells. Following subcutaneous injection of these cells into nude mice, fibrosarcoma were formed from SLC30A1- or SERPINB2-expressing cells. Reduced plasma levels of miR-182 were detected in subjects exposed to high level of PM2.5 than in those exposed to low level of PM2.5 (P = 0.043). Similar results were seen for miR-185 although the difference was not statistically significant (P = 0.328). Increased expressions of SLC30A1, SERPINB2 and AKR1C1 were detected in human lung cancer. These results suggest that modulation of miR-182 and miR-185 and their target genes may contribute to lung carcinogenesis attributable to PM2.5 exposure. PMID:26338969

  2. The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

    PubMed

    Bittencourt, L S; Machado, D C; Machado, M M; Dos Santos, G F F; Algarve, T D; Marinowic, D R; Ribeiro, E E; Soares, F A A; Barbisan, F; Athayde, M L; Cruz, I B M

    2013-03-01

    The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 μM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels. PMID:23220610

  3. A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

    PubMed Central

    Doherty, F J; Wassell, J A; Mayer, R J

    1987-01-01

    Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Images Fig. 1. Fig. 5. PMID:3593223

  4. Improved biolistic transfection of hair cells.

    PubMed

    Zhao, Hongyu; Avenarius, Matthew R; Gillespie, Peter G

    2012-01-01

    Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. PMID:23049715

  5. Improved Biolistic Transfection of Hair Cells

    PubMed Central

    Gillespie, Peter G.

    2012-01-01

    Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca2+-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. PMID:23049715

  6. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  7. Sulfonate groups grafted on Ti6Al4V favor MC3T3-E1 cell performance in serum free medium conditions.

    PubMed

    Felgueiras, Helena; Migonney, Véronique

    2014-06-01

    Ten years ago, we synthesized "bioactive model polymers" bearing sulfonate groups and proposed a mechanism of their modulation effect at different steps of the cell response. Then, we set up the grafting of polymers bearing sulfonate on Ti6Al4V surfaces by a grafting "from" technique making sure of the creation of covalent bonds between the grafted polymer and the Ti6Al4V surface. We have checked and confirmed the positive effect of grafted sulfonate groups on the osteoblastic cell response in vivo and in vitro but we did not elucidate the mechanism. The aim of this basic work consists first in investigating the role of sulfonate groups in the presence and in the absence of proteins at early stages of the osteointegration process on poly(sodium styrene sulfonate) poly(NaSS) grafted and ungrafted Ti6Al4V surfaces, in vitro. To understand the role of poly(NaSS) grafted chains on osteoblast-like cell response and to confirm/elucidate the importance of fetal bovine serum (FBS) proteins in the culture medium, MC3T3-E1 cells were seeded onto poly(NaSS) grafted and non-grafted Ti6Al4V surfaces. Cultures were carried out in a complete (10% FBS) and in a non-complete medium (without FBS). Cell viability assay, cell attachment number and cell adhesion strength were followed up to 3days of culture. The presence of proteins enhanced cell growth and development whatever the surface and the presence of sulfonate groups enhanced the cell attachment even in the absence of proteins, which suggests and confirms that the sulfonate groups can modify the activity of cells such as the secretion of binding proteins. Statistical differences were found in the attachment strength tests on poly(NaSS) grafted and ungrafted surfaces and showed that the sulfonate groups play an important role in the cell resistance to shear stress. PMID:24863216

  8. Rubus coreanus Miq. extract promotes osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells.

    PubMed

    Lee, Kyung-Hee; Choi, Eun-Mi

    2006-01-01

    To prevent bone loss that occurs with increasing age, certain nutritional and pharmacological factors are needed. In the present study, the ethanol extract from the fruit of Rubus coreanus Miq. (RCE) was investigated for its effect on the function of osteoblastic MC3T3-E1 cells. RCE (10approximately50 microg/ml) caused a significant elevation in cell viability, alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in the cells. The effect of RCE (50 microg/ml) in increasing cell viability, ALP activity, and collagen content was prevented by the presence of 10(-6) M cycloheximide and 10(-6) M tamoxifen, suggesting that RCE's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of RCE on the H(2)O(2)-induced apoptosis and production of local factors in osteoblasts. Treatment with RCE (10approximately50 microg/ml) decreased the 0.2 mM H(2)O(2)-induced apoptosis and production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and nitric oxide (NO) in osteoblasts. Our data indicate that the enhancement of osteoblast function by Rubus coreanus Miq. may result in the prevention of osteoporosis and inflammatory bone diseases. PMID:16883635

  9. Nonivamide Enhances miRNA let‐7d Expression and Decreases Adipogenesis PPARγ Expression in 3T3‐L1 Cells

    PubMed Central

    Rohm, Barbara; Holik, Ann‐Katrin; Kretschy, Nicole; Somoza, Mark M.; Ley, Jakob P.; Widder, Sabine; Krammer, Gerhard E.; Marko, Doris

    2015-01-01

    ABSTRACT Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti‐obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP‐analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3‐L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans‐tert‐butylcyclohexanol revealed that the anti‐adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro‐adipogenic transcription factor peroxisome‐proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu‐let‐7d‐5p, which has been associated with decreased PPARγ levels. J. Cell. Biochem. 116: 1153–1163, 2015. © 2015 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. PMID:25704235

  10. Differences in the bone differentiation properties of MC3T3-E1 cells on polished bulk and sputter-deposited titanium specimens.

    PubMed

    Oya, Kei; Tanaka, Yuta; Moriyama, Yoshihisa; Yoshioka, Yuki; Kimura, Tsuyoshi; Tsutsumi, Yusuke; Doi, Hisashi; Nomura, Naoyuki; Noda, Kazuhiko; Kishida, Akio; Hanawa, Takao

    2010-08-01

    The roughness and cleanness of a titanium surface must be controlled in order to investigate the expression mechanism of hard tissue compatibility on titanium. In this study, osteogenic MC3T3-E1 cells were cultured and differentiation-induced on bulk and sputter-deposited titanium specimens, and the osteogenesis were investigated. For the preparation of bulk specimens, titanium discs were mirror-polished. On the other hand, titanium was sputter-deposited on smooth and clean cover glasses as sputter-deposited specimens. As a result, no significant difference was observed in the cell morphology and attached number. On the other hand, the time showing maximum activity in the alkaline phosphatase and gene expressions, which are related to bone differentiation on the bulk titanium, were superior to those on the sputter-deposited titanium. From the surface observation of the specimens with a scanning electron microscope and a scanning probe microscope, the surface on the sputter-deposited titanium was more uniform and cleaner than that on the bulk titanium. According to X-ray photoelectron spectroscopy, the thickness of surface oxide film on the sputter-deposited titanium was smaller than that on the bulk titanium. In addition, the proportions of TiO and Ti(2)O(3) in the surface oxide film on the sputter-deposited titanium were larger than those on the bulk titanium. These differences might influence the differentiation of osteoblastic cells. PMID:20198694

  11. Distinctive gene expression profiles in Balb/3T3 cells exposed to low dose cobalt nanoparticles, microparticles and ions: potential nanotoxicological relevance.

    PubMed

    Perconti, S; Aceto, G M; Verginelli, F; Napolitano, F; Petrarca, C; Bernardini, G; Raiconi, G; Tagliaferri, R; Sabbioni, E; Di Gioacchino, M; Mariani-Costantini, R

    2013-01-01

    Size-dependent characteristics of novel engineered nanomaterials might result in unforeseen biological responses and toxicity. To address this issue, we used cDNA microarray analysis (13443 genes) coupled with bioinformatics and functional gene annotation studies to investigate the transcriptional profiles of Balb/3T3 cells exposed to a low dose (1 μM) of cobalt nanoparticles (CoNP), microparticles (CoMP) and ions (Co2+). CoNP, CoMP and Co2+ affected 124, 91 and 80 genes, respectively. Hierarchical clustering revealed two main gene clusters, one up-regulated, mainly after Co2+, the other down-regulated, mainly after CoNP and CoMP. The significant Gene Ontology (GO) terms included oxygen binding and transport and hemoglobin binding for Co2+, while the GOs of CoMP and CoNP were related to nucleus and intracellular components. Pathway analysis highlighted: i) mitochondrial dysfunction for Co2+, ii) signaling, activation of innate immunity, and apoptosis for CoNP, and iii) cell metabolism, G1/S cell cycle checkpoint regulation and signaling for CoMP. Unlike ions, particles affected toxicologically-relevant pathways implicated in carcinogenesis and inflammation. PMID:23830394

  12. α-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 Cells via PPARγ, GLUT4, and Leptin Expressions

    PubMed Central

    Taher, Muhammad; Mohamed Amiroudine, Mohamed Zaffar Ali; Tengku Zakaria, Tengku Muhamad Faris Syafiq; Ichwan, Solachuddin J. A.; Kaderi, Mohd Arifin; Ahmed, Qamar Uddin; Zakaria, Zainul Amiruddin

    2015-01-01

    Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 diabetes, and cancer. The development of obesity is also accompanied by significant differentiation of preadipocytes into adipocytes. In this study, we investigated the activity of α-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and adipocyte differentiation of 3T3-L1 cells focusing on PPARγ, GLUT4, and leptin expressions. α-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50 μM of α-mangostin reduced intracellular fat accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[3H] glucose uptake activity showed that α-mangostin significantly improved the glucose uptake (P < 0.05) with highest activity found at 25 μM. In addition, α-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50 μM of α-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPARγ gene expression. Induction of glucose uptake and free fatty acid release by α-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that α-mangostin might be possible candidate for the effective management of obesity in future. PMID:25873982

  13. Technical modification of the Balb/c 3T3 cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol.

    PubMed

    Hayashi, Kumiko; Sasaki, Kiyoshi; Asada, Shin; Tsuchiya, Toshiyuki; Hayashi, Makoto; Yoshimura, Isao; Tanaka, Noriho; Umeda, Makoto

    2008-12-01

    The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2microg/ml insulin, rather than the more-complex insulin-transferrin-ethanolamine-sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24-25 days to 31-32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl(2) and phenacetin, combining a post-treatment of 100ng/ml 12-O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl(2) and phenobarbital, with pre-treatment of 0.2microg/ml MCA at the initiation stage. In the present study, only

  14. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways.

    PubMed

    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen; Yayi, Xia

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. PMID:27060196

  15. Viral-cellular junction fragment from a human papillomavirus type 16-positive tumor is competent in transformation of NIH 3T3 cells

    SciTech Connect

    Le, J.Y.; Defendi, V.

    1988-11-01

    A 4.4-kilobase DNA fragment (T4.4) from a human tumor was found to be competent to fully transform NIH 3T3 cells. This competency resides in the whole hybrid DNA fragment, since the separate viral or cellular DNA sequences were not active. Abundant E6-E7 transcripts were found in the transformed cells. When the cellular fragments were substituted with polyadenylation sequences from polyomavirus or simian virus 40 DNA, little or no restoration of transforming activity was observed. In experiments in which an exogenous reporting gene, that for chloramphenicol acetyltransferase, was used, the possibility was excluded that the cellular flanking sequences act as a traditional enhancer; yet, when the cellular sequences were placed downstream of a cloramphenicol acetyltransferase expression vector (pSV2 CAT), activity of the reference gene was clearly enhanced. These results indicate that DNA containing human papillomavirus type 16 open reading frames E6 and E7 isolated from the genome of a human tumor has transforming potential, but this potential is realized when the viral DNA is joined to cellular sequences, and that the cellular sequences function in a more complex way than by simply providing polyadenylation signals.

  16. HSP110 expression is induced by cadmium exposure but is dispensable for cell survival of mouse NIH3T3 fibroblasts.

    PubMed

    Ridley, Wakako; Nishitai, Gen; Matsuoka, Masato

    2010-05-01

    The effects of cadmium exposure on the expression of HSP110 were examined in mouse NIH3T3 fibroblasts. Following exposure to cadmium chloride, the level of HSP110 and HSP70 proteins increased after 3h and remained elevated at 24h. Similarly, their mRNA levels increased markedly in response to cadmium exposure. Treatment with 10μM mercury chloride, another toxic metal compound, also induced expression of HSP110; however, HSP110 expression was not induced in cells exposed to the same concentration of manganese chloride, zinc chloride, or lead chloride for 6 or 24h. Silencing of HSP110 expression using short-interference RNA did not affect cadmium-induced cellular damage. These results show that cadmium exposure induces the expression of high molecular weight chaperone HSP110 as well as the well-known HSP70, but indicate that HSP110 does not play a major role in cell survival following cadmium exposure. PMID:21787611

  17. In vitro and in vivo enhancement of adipogenesis by Italian ryegrass (Lolium multiflorum) in 3T3-L1 cells and mice.

    PubMed

    Valan Arasu, Mariadhas; Ilavenil, Soundharrajan; Kim, Da Hye; Gun Roh, Sang; Lee, Jeong-Chae; Choi, Ki Choon

    2014-01-01

    Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as α-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-γ2, CCAAT/enhancer binding protein-α, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPARγ-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals. PMID:24454838

  18. In Vitro and In Vivo Enhancement of Adipogenesis by Italian Ryegrass (Lolium multiflorum) in 3T3-L1 Cells and Mice

    PubMed Central

    Kim, Da Hye; Gun Roh, Sang; Lee, Jeong-Chae; Choi, Ki Choon

    2014-01-01

    Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as α-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-γ2, CCAAT/enhancer binding protein-α, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPARγ-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals. PMID:24454838

  19. In Vitro Selective Anti-Proliferative Effect of Zinc Oxide Nanoparticles Against Co-Cultured C2C12 Myoblastoma Cancer and 3T3-L1 Normal Cells.

    PubMed

    Chandrasekaran, Murugesan; Pandurangan, Muthuraman

    2016-07-01

    The zinc oxide (ZnO) nanoparticle has been widely used in biomedical applications and cancer therapy and has been reported to induce a selective cytotoxic effect on cancer cell proliferation. The present study investigated the cytotoxicity of ZnO nanoparticles against co-cultured C2C12 myoblastoma cancer cells and 3T3-L1 adipocytes. Our results showed that the ZnO nanoparticles could be cytotoxic to C2C12 myoblastoma cancer cells than 3T3-L1 cells. The messenger RNA (mRNA) expressions of p53 and bax were significantly increased 114.3 and 118.2 % in the C2C12 cells, whereas 42.5 and 40 % were increased in 3T3-L1 cells, respectively. The mRNA expression of bcl-2 was reduced 38.2 and 28.5 % in the C2C12 and 3T3-L1 cells, respectively, whereas the mRNA expression of caspase-3 was increased 80.7 and 51.6 % in the C2C12 and 3T3-L1 cells, respectively. The protein expressions of p53, bax, and caspase-3 were significantly increased 40, 81.8, and 80 % in C2C12 cells, whereas 20.3, 28.2, and 37.9 % were increased in 3T3-L1 cells, respectively. The mRNA expression of bcl-2 was significantly reduced 32.2 and 22.7 % in C2C12 and 3T3-L1 cells, respectively. Caspase-3 enzyme activity and reactive oxygen species (ROS) were increased in co-cultured C2C12 cells compared to 3T3-L1 cells. Taking all these data together, it may suggest that ZnO nanoparticles severely induce apoptosis in C2C12 myoblastoma cancer cells than 3T3-L1 cells. PMID:26563419

  20. A panel of in vitro tests to evaluate genotoxic and morphological neoplastic transformation potential on Balb/3T3 cells by pristine and remediated titania and zirconia nanoparticles.

    PubMed

    Stoccoro, Andrea; Di Bucchianico, Sebastiano; Uboldi, Chiara; Coppedè, Fabio; Ponti, Jessica; Placidi, Claudia; Blosi, Magda; Ortelli, Simona; Costa, Anna Luisa; Migliore, Lucia

    2016-09-01

    The FP7 Sanowork project was aimed to minimise occupational hazard and exposure to engineered nanomaterials (ENM) through the surface modification in order to prevent possible health effects. In this frame, a number of nanoparticles (NP) have been selected, among which zirconium (ZrO2) and titanium (TiO2) dioxide. In this study, we tested ZrO2 NP and TiO2 NP either in their pristine (uncoated) form, or modified with citrate and/or silica on their surface. As benchmark material, Aeroxide® P25 was used. We assessed cytotoxicity, genotoxicity and induction of morphological neoplastic transformation of NP by using a panel of in vitro assays in an established mammalian cell line of murine origin (Balb/3T3). Cell viability was evaluated by means of colony-forming efficiency assay (CFE). Genotoxicity was investigated by cytokinesis-block micronucleus cytome assay (CBMN cyt) and comet assay, and by the use of the restriction enzymes EndoIII and Fpg, oxidatively damaged DNA was detected; finally, the morphological neoplastic transformation of NP was assayed in vitro by cell transformation assay (CTA). Our results show that the surface remediation has not been effective in modifying cyto- and genotoxic properties of the nanomaterials tested; indeed, in the case of remediation of zirconia and titania with citrate, there is a tendency to emphasise the toxic effects. The use of a panel of assays, such as those we have employed, allowing the evaluation of multiple endpoints, including cell transformation, seems particularly advisable especially in the case of long-term exposure effects in the same cell type. PMID:27056944

  1. Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPARγ2 Splicing during Adipogenesis in 3T3-L1 Cells

    PubMed Central

    Cooper, Denise R.; Carter, Gay; Li, Pengfei; Patel, Rehka; Watson, James E.; Patel, Niketa A.

    2014-01-01

    Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPARγ mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPARγ2 variant was spliced and when terminal differentiation occurs The appearance of PPARγ2 coordinates with the PPARγ1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPARγ1 and, primarily, PPARγ2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPARγ2, but not γ1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPARγ2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events. PMID:25437750

  2. Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

    PubMed Central

    Dessaux, Sophie; Besse, Anthony; Brahimi-Adouane, Sabrina; Emile, Jean-François; Blay, Jean-Yves; Alberti, Laurent

    2013-01-01

    Objective Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations. Materials and Methods Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples. Results Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway. Conclusion Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways. PMID:23593401

  3. Traditional medicine yanggyuksanhwa-tang inhibits adipogenesis and suppresses proliferator-activated receptor-gamma expression in 3T3-L1 cells

    PubMed Central

    Jeong, Soo-Jin; Yoo, Sae-Rom; Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Background: Yanggyuksanhwa-tang (YGSHT) is a specific traditional Korean herbal formula for Soyangin according to Sasang constitutional philosophy. Although its biological activities against inflammation and cerebral infarction have been reporting, there is no information about the adipogenic activity of YGSHT. In the present study, we investigated the anti-adipogenic activity of YGSHT to evaluate effects of YGSHT on adipogenesis in vitro. Materials and Methods: Using 3T3-L1 preadipocytes, we induced the cellular differentiation into adipocytes by adding insulin. Anti-adipogenic activity of YGSHT was measured by oil red O staining, triglyceride assay, glycerol-3-phosphate dehydrogenase (GPDH) activity test, and leptin assay. Results: YGSHT extract had no significant cytotoxicity in preadipocytes or differentiated adipocytes. YGSHT reduced the number of lipid droplets and content of triglyceride in adipose cells. YGSHT also significantly inhibited GPDH activity and decreased leptin production compared with control adipocytes. Down-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression at the messenger RNA level was observed in YGSHT-treated adipocytes. Conclusion: Taken together, our data suggest that YGSHT has potential as an anti-obesity drug candidate. PMID:26246724

  4. Platycodin D inhibits lipogenesis through AMPKα-PPARγ2 in 3T3-L1 cells and modulates fat accumulation in obese mice.

    PubMed

    Lee, Eun Jeong; Kang, Minseok; Kim, Yeong Shik

    2012-09-01

    Platycodin D (PD) has been reported to control obesity in vivo. This study investigated the molecular mechanism of PD, focusing on its ability to decrease the expression of adipogenic factors through AMP-activated protein kinase α (AMPKα) in adipocytes and its ability to prevent abdominal fat accumulation in high-fat diet-induced obese C57BL/6 mice. The inhibitory effect of lipid accumulation in 3T3-L1 cells was measured by Oil Red O staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting. To determine the antiobesity effect in vivo, one group of mice were given a normal diet and the others were fed a high-fat diet for 8 weeks. The high-fat diet mice were then assigned to one of three subgroups: aminoimidazole carboxamide ribonucleotide (AICAR), vehicle, and PD. PD significantly reduced fat accumulation by inhibiting adipogenic signal transcriptional factors, such as peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPα), which functions via AMPK signaling, in vitro. PD reduced both body weight and fat volume; consequently, lipid metabolism was improved by increasing AMPKα, similar to AICAR, and reduced PPARγ2 and C/EBPα expression in adipose tissue. The results suggested that PD could be used to decrease the expression of adipogenic factors related to the AMPK pathway. Hence, PD could be an alternative treatment for controlling obesity by downregulating lipid accumulation. PMID:22872592

  5. Platycodon grandiflorum A. De Candolle Ethanolic Extract Inhibits Adipogenic Regulators in 3T3-L1 Cells and Induces Mitochondrial Biogenesis in Primary Brown Preadipocytes.

    PubMed

    Kim, Hye-Lin; Park, Jinbong; Park, Hyewon; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Jeong, Mi-Young; Um, Jae-Young

    2015-09-01

    This study was designed to evaluate the effects of Platycodon grandiflorum A. DC. ethanolic extract (PG) on obesity in brown/white preadipocytes. The effect of PG on the differentiation and mitochondrial biogenesis of brown adipocytes is still not examined. An in vivo study showed that PG induced weight loss in mice with high-fat-diet-induced obesity. PG successfully suppressed the differentiation of 3T3-L1 cells by down-regulating cellular induction of the peroxisome proliferators activated receptor γ (PPARγ), CCAAT enhancer binding protein α (C/EBPα), lipin-1, and adiponectin but increasing expression of silent mating type information regulation 2 homologue 1 (SIRT1) and the phosphorylation of AMP-activated protein kinase α (AMPKα). The effect of PG on the adipogenic factors was compared with that of its bioactive compound platycodin D. In addition, PG increased expressions of mitochondria-related genes, including uncoupling protein 1 (UCP1), peroxisome proliferator activated receptor-coactivator 1 α (PGC1α), PR domain containing 16 (PRDM16), SIRT3, nuclear respiratory factor (NRF), and cytochrome C (CytC) in primary brown adipocytes. These results indicate that PG stimulates the differentiation of brown adipocytes through modulation of mitochondria-related genes and could offer clinical benefits as a supplement to treat obesity. PMID:26244589

  6. Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

    PubMed Central

    Karasawa, Yoko; Tanaka, Hideki; Nakai, Kumiko; Tanabe, Natsuko; Kawato, Takayuki; Maeno, Masao; Shimizu, Noriyoshi

    2015-01-01

    Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways. Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting. Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3. Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts. PMID:26640410

  7. Proliferation and osteogenic response of MC3T3-E1 pre-osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria.

    PubMed

    Hadjicharalambous, Chrystalleni; Mygdali, Evdokia; Prymak, Oleg; Buyakov, Ales; Kulkov, Sergei; Chatzinikolaidou, Maria

    2015-11-01

    Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix. PMID:25847599

  8. Nanoparticles of compacted DNA transfect postmitotic cells.

    PubMed

    Liu, Ge; Li, DeShan; Pasumarthy, Murali K; Kowalczyk, Tomasz H; Gedeon, Christopher R; Hyatt, Susannah L; Payne, Jennifer M; Miller, Timothy J; Brunovskis, Peter; Fink, Tamara L; Muhammad, Osman; Moen, Robert C; Hanson, Richard W; Cooper, Mark J

    2003-08-29

    Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore. PMID:12807905

  9. Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells.

    PubMed

    Alcantara, Ethel H; Shin, Mee-Young; Sohn, Ho-Yong; Park, Youn-Moon; Kim, Taewan; Lim, Jae-Hwan; Jeong, Hyung-Jin; Kwon, Soon-Tae; Kwun, In-Sook

    2011-11-01

    Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation. PMID:21292464

  10. Resveratrol inhibits lipogenesis of 3T3-L1 and SGBS cells by inhibition of insulin signaling and mitochondrial mass increase.

    PubMed

    Li, Shuijie; Bouzar, Célia; Cottet-Rousselle, Cécile; Zagotta, Ivana; Lamarche, Frédéric; Wabitsch, Martin; Tokarska-Schlattner, Malgorzata; Fischer-Posovszky, Pamela; Schlattner, Uwe; Rousseau, Denis

    2016-06-01

    Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans. PMID:26968895

  11. Sodium alginate-cross-linked polymyxin B sulphate-loaded solid lipid nanoparticles: Antibiotic resistance tests and HaCat and NIH/3T3 cell viability studies.

    PubMed

    Severino, Patrícia; Chaud, Marco V; Shimojo, Andrea; Antonini, Danilo; Lancelloti, Marcelo; Santana, Maria Helena A; Souto, Eliana B

    2015-05-01

    Polymyxins are a group of antibiotics with a common structure of a cyclic peptide with a long hydrophobic tail. Polymyxin B sulphate (PLX) has cationic charge, which is an obstacle for the efficient loading into Solid Lipid Nanoparticles (SLN). In the present paper, we describe an innovative method to load PLX into SLN to achieve the sustained release of the drug. PLX was firstly cross-linked with sodium alginate (SA) at different ratios (1:1, 1:2 and 1:3 SA/PLX), and loaded into SLN produced by high pressure homogenization (HPH). Optimized SLN were produced applying 500bar pressure and 5 homogenization cycles. The best results were obtained with SA/PLX (1:1), recording 99.08±1.2% for the association efficiency of the drug with SA, 0.99±10g for the loading capacity and 212.07±5.84% degree of swelling. The rheological profile of aqueous SA solution followed the typical behaviour of concentrated polymeric solutions, whereas aqueous SA/PLX solution exhibited a gel-like dynamic behaviour. Micrographs show that SA/PLX depicted a porous and discontinuous amorphous phase in different ratios. The encapsulation efficiency of SA/PLX (1:1) in SLN, the mean particle diameter, polydispersity index and zeta potential were, respectively, 82.7±5.5%; 439.5±20.42nm, 0.241±0.050 and -34.8±0.55mV. The effect of SLN on cell viability was checked in HaCat and NIH/3T3 cell lines, and the minimal inhibitory concentrations (MIC) were determined in Pseudomonas aeruginosa strains. SA/PLX-loaded SLN were shown to be less toxic than free PLX. Minimal inhibitory concentrations (MIC) showed the presence of the cross-linker polymer-drug complex, and SLN were shown to enhance MIC in the evaluated strains. PMID:25863712

  12. Effect of fluoride on insulin level of rats and insulin receptor expression in the MC3T3-E1 cells.

    PubMed

    Hu, Chun-Yan; Ren, Li-Qun; Li, Xi-Ning; Wu, Nan; Li, Guang-Sheng; Liu, Qin-Yi; Xu, Hui

    2012-12-01

    Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis. PMID:22872571

  13. Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells.

    PubMed

    Takeuchi, T; Tsuboi, T; Arai, M; Togari, A

    2001-03-01

    It is well known that adrenergic agonists efficiently activate beta-adrenoceptors on osteoblastic cells and can stimulate bone resorption in intact mouse calvaria. Recently, an osteoclastogenic factor of osteoblastic origin was found to be a novel tumor necrosis factor ligand family member and was termed osteoclast differentiation factor (ODF). Using a reverse transcription-polymerase chain reaction approach, we investigated the effect of epinephrine on mRNA levels of ODF and its decoy receptor, osteoclastogenesis inhibitory factor (OCIF), in MC3T3-E1 cells. Treatment with epinephrine (1 microM) rapidly increased ODF and OCIF mRNA levels, which peaked after 0.5 hr of treatment. Epinephrine (1 microM) also increased interleukin (IL)-6, IL-11, and cyclooxygenase (COX)-II mRNA levels, as well as increased prostaglandin E(2) (PGE(2)) accumulation in the culture medium. Treatment of the cells with IL-11 (10 ng/mL) or PGE(2) (1 microM) increased ODF and OCIF mRNA levels as observed with epinephrine. However, increases in ODF and OCIF mRNA levels by epinephrine were more rapid than those by IL-11, and were not influenced by NS-398 (100 microM; an inhibitor of COX-II), suggesting a direct effect of epinephrine on ODF and OCIF mRNA expressions as well as an indirect effect mediated by IL-11 and PGE(2) production. Epinephrine-induced increases in ODF and OCIF mRNA levels were inhibited by pretreatment with timolol (1 microM; beta-antagonist) and phentolamine (1 microM; alpha-antagonist), respectively. Furthermore, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells from mouse bone marrow cells was stimulated by isoproterenol (0.1 to 10 microM) or epinephrine (0.1 to 10 microM). The action of isoproterenol, a beta-agonist, was clearly stronger than that of epinephrine, suggesting the importance of the physiological balance between ODF and OCIF productions for osteoclastogenesis. These findings suggest that beta-adrenergic stimulation induces not

  14. Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate

    PubMed Central

    Romero, María del Mar; Sabater, David; Fernández-López, José Antonio; Remesar, Xavier; Alemany, Marià

    2015-01-01

    Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic

  15. Effects and mechanisms of 8-prenylnaringenin on osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7

    PubMed Central

    Luo, Dan; Kang, Lumei; Ma, Yuhui; Chen, Hongping; Kuang, Haibin; Huang, Qiren; He, Ming; Peng, Weijie

    2014-01-01

    8-Prenylnaringenin (8-PN) is a phytoestrogen with the highest estrogenic activity. The objective of the present study was to confirm the superiority of 8-PN on bone metabolisms and the estrogen receptor (ER) subtype mediating effects of 8-PN. The osteoblast MC3T3-E1 and osteoclast-like cell line RAW264.7 were treated with 17β-estradiol (10−8 mol/L), genistein (10−5 mol/L), daidzein (10−5 mol/L), 8-PN (10−5 mol/L) alone or in the presence of ERα antagonist MPP (10−7 mol/L) and ERβ antagonist PTHPP (1.5 × 10−7 mol/L). It has been found that 8-PN did not affect osteoblast proliferation, and that 8-PN increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) concentrations, and the mineralized nodules. 8-PN inhibited RAW264.7 differentiating into osteoclasts and reduced the pit area of bone resorption. 8-PN could also inhibit the protein and mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts, and conversely promote the expression of osteoprotegerin (OPG). These effects of 8-PN were mainly inhibited not by PTHPP but by MPP and they were weaker than estrogen's effects but stronger than those of genistein and daidzein. In conclusion, the effects of 8-PN on promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption were mediated by ERα instead of ERβ and the efficacy was more potent than that of the two classic phytoestrogens: genistein and daidzein. PMID:25473491

  16. Effects and mechanisms of 8-prenylnaringenin on osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7.

    PubMed

    Luo, Dan; Kang, Lumei; Ma, Yuhui; Chen, Hongping; Kuang, Haibin; Huang, Qiren; He, Ming; Peng, Weijie

    2014-07-01

    8-Prenylnaringenin (8-PN) is a phytoestrogen with the highest estrogenic activity. The objective of the present study was to confirm the superiority of 8-PN on bone metabolisms and the estrogen receptor (ER) subtype mediating effects of 8-PN. The osteoblast MC3T3-E1 and osteoclast-like cell line RAW264.7 were treated with 17β-estradiol (10(-8) mol/L), genistein (10(-5) mol/L), daidzein (10(-5) mol/L), 8-PN (10(-5) mol/L) alone or in the presence of ERα antagonist MPP (10(-7) mol/L) and ERβ antagonist PTHPP (1.5 × 10(-7) mol/L). It has been found that 8-PN did not affect osteoblast proliferation, and that 8-PN increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) concentrations, and the mineralized nodules. 8-PN inhibited RAW264.7 differentiating into osteoclasts and reduced the pit area of bone resorption. 8-PN could also inhibit the protein and mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts, and conversely promote the expression of osteoprotegerin (OPG). These effects of 8-PN were mainly inhibited not by PTHPP but by MPP and they were weaker than estrogen's effects but stronger than those of genistein and daidzein. In conclusion, the effects of 8-PN on promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption were mediated by ERα instead of ERβ and the efficacy was more potent than that of the two classic phytoestrogens: genistein and daidzein. PMID:25473491

  17. Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

    PubMed

    Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

    2005-12-01

    Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions. PMID:16274879

  18. Flavonol acylglycosides from flower of Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells.

    PubMed

    Yahagi, Tadahiro; Daikonya, Akihiro; Kitanaka, Susumu

    2012-01-01

    Obesity is a serious health problem worldwide. We investigated the anti-obesity effect of the flower of Albizia julibrissin DURAZZ. (Leguminosae). A 90% EtOH extract of the flower inhibited adipogenesis in 3T3-L1 preadipocytes, as well as the activity of glycerol-3-phosphate dehydrogenase (GPDH) activity. New flavonol acylglycosides (1-4) and eighteen known compounds (5-22) were isolated by bioassay-directed fractionation. These new glycosides were elucidated to be 3″-(E)-p-coumaroylquercitrin (1), 3″-(E)-feruloylquercitrin (2), 3″-(E)-cinnamoylquercitrin (3), and 2″-(E)-cinnamoylquercitrin (4) on the basis of spectroscopic and chemical analysis. These compounds inhibited adipogenesis in 3T3-L1 preadipocytes. In particular, 2 exhibited potent inhibitory effects on triglyceride accumulation. Furthermore, GPDH activity was inhibited by 2. Additionally, 2 inhibited glucose uptake in 3T3-L1 adipocytes. These results indicate that the 90% EtOH extract and compounds isolated from the flower of A. julibrissin inhibit adipogenesis in 3T3-L1 preadipocytes and may have anti-obesity effect through the inhibition of preadipocyte differentiation. PMID:22223384

  19. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    NASA Astrophysics Data System (ADS)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  20. Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function.

    PubMed

    Waldman, Maayan; Bellner, Lars; Vanella, Luca; Schragenheim, Joseph; Sodhi, Komal; Singh, Shailendra P; Lin, Daohong; Lakhkar, Anand; Li, Jiangwei; Hochhauser, Edith; Arad, Michael; Darzynkiewicz, Zbigniew; Kappas, Atallah; Abraham, Nader G

    2016-07-15

    Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1α and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1α as cells deficient in PGC-1α (PGC-1α knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1α-dependent manner, P < 0.05. The increase in PGC-1α was associated with an increase in β-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1α is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1α signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation. PMID:27224420

  1. Regulation of heme oxygenase-1 mRNA deadenylation and turnover in NIH3T3 cells by nitrosative or alkylation stress

    PubMed Central

    Leautaud, Veronica; Demple, Bruce

    2007-01-01

    Background Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most sensitive indicators of cellular stress. Previous work in human fibroblasts has shown that HO-1 expression is induced by NO, and that transcriptional induction is only partially responsible; instead, the HO-1 mRNA half-life is substantially increased in response to NO. The mechanism of this stabilization remains unknown. Results In NIH3T3 murine fibroblasts, NO exposure increased the half-life of the HO-1 transcript from ~1.6 h to 11 h, while treatments with CdCl2, NaAsO2 or H2O2 increased the half-life only up to 5 h. Although poly(A) tail shortening can be rate-limiting in mRNA degradation, the HO-1 mRNA deadenylation rate in NO-treated cells was ~65% of that in untreated controls. In untreated cells, HO-1 poly(A) removal proceeded until 30–50 nt remained, followed by rapid mRNA decay. In NO-treated cells, HO-1 deadenylation stopped with the mRNA retaining poly(A) tails 30–50 nt long. We hypothesize that NO treatment stops poly(A) tail shortening at the critical 30- to 50-nt length. This is not a general mechanism for the post-transcriptional regulation of HO-1 mRNA. Methyl methane sulfonate also stabilized HO-1 mRNA, but that was associated with an 8-fold decrease in the deadenylation rate compared to that of untreated cells. Another HO-1 inducer, CdCl2, caused a strong increase in the mRNA level without affecting the HO-1 mRNA half-life. Conclusion The regulation of HO-1 mRNA levels in response to cellular stress can be induced by transcriptional and different post-transcriptional events that act independently, and vary depending on the stress inducer. While NO appears to stabilize HO-1 mRNA by preventing the final steps of deadenylation, methyl methane sulfonate achieves stabilization through the regulation of earlier stages of deadenylation. PMID:18096048

  2. Transformation of BALB/c-3T3 cells: V. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays.

    PubMed Central

    Matthews, E J; Spalding, J W; Tennant, R W

    1993-01-01

    This report describes the activities of 168 chemicals tested in a standard transformation assay using A-31-1-13 BALB/c-3T3 cells. The data set includes 84 carcinogens, 77 noncarcinogens, and 7 research chemicals. Carcinogens included 49 mutagens and 35 nonmutagens; noncarcinogens included 24 mutagens and 53 nonmutagens. The transformation assay did not use an exogenous activation system, thus, all chemical responses depended on the inherent target cell metabolic capacity where metabolic activation was required. The upper dose limit was 100 milli-osmolar because the assay could not discriminate active and inactive chemicals tested above this concentration. Certain physicochemical properties resulted in technical problems that affected chemical biological activity. For example, chemicals that reacted with plastic were usually nonmutagenic carcinogens. Similarly, chemicals that were insoluble in medium, or bound metals, were usually nonmutagenic and nontransforming. Multifactorial data analyses revealed that the transformation assay discriminated between nonmutagenic carcinogens and noncarcinogens; it detected 64% of the carcinogens and only 26% of the noncarcinogens. In contrast, the transformation assay detected most mutagenic chemicals, including 94% of the mutagenic carcinogens and 70% of the mutagenic noncarcinogens. Thus, transformation or Salmonella typuimurium mutagenicity assays could not discriminate mutagenic carcinogens from mutagenic noncarcinogens. Data analyses also revealed that mutagenic chemicals were more cytotoxic than nonmutagenic chemicals; 88% of the mutagens had an LD50 < 5 mM, whereas half of the nonmutagens had an LD50 > 5 mM. Binary data analyses of the same data set revealed that the transformation assay and rodent bioassay had a concordance of 71%, a sensitivity for carcinogens of 80.0%, and a specificity for detecting noncarcinogens of 60%. In contrast, Salmonella mutagenicity assays and rodent bioassays had a concordance of 63%, a

  3. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. )

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  4. 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine β-synthase

    PubMed Central

    Kriebitzsch, Carsten; Verlinden, Lieve; Eelen, Guy; van Schoor, Natasja M.; Swart, Karin; Lips, Paul; Meyer, Mark B.; Pike, J Wesley; Boonen, Steven; Carlberg, Carsten; Vitvitsky, Victor; Bouillon, Roger; Banerjee, Ruma; Verstuyf, Annemieke

    2011-01-01

    High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY due to its negative impact on collagen maturation. HCY is cleared by cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine pre-osteoblasts treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)2D3. Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)2D3 treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)2D3-treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knock-out osteoblasts, in which up-regulation of cbs in response to 1,25(OH)2D3 was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D3 levels] and HCY levels. In conclusion, this study demonstrated that cbs is a primary 1,25(OH)2D3 target gene which renders HCY metabolism responsive to 1,25(OH)2D3. PMID:21898591

  5. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  6. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents.

    PubMed

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-07-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  7. Early events elicited by Bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

    SciTech Connect

    Zachary, I.; Sinnett-Smith, J.W.; Rozengurt, E.

    1986-01-01

    Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an M/sub r/ 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent. The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of /sup 125/I-labeled epidermal growth factor (/sup 125/I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca/sup 2 +/ concentration does not mediate the bombesin inhibition of /sup 125/I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of /sup 125/I-EGF to its cellular receptor.

  8. Glyoxalase I in detoxification: studies using a glyoxalase I transfectant cell line.

    PubMed Central

    Ranganathan, S; Walsh, E S; Tew, K D

    1995-01-01

    The glyoxalase system (glyoxalase I, glyoxalase II and GSH as cofactor) is involved in the detoxification of methylglyoxal (a byproduct of the glycolytic pathway) and other alpha-oxoaldehydes. We have transfected a 622 bp cDNA encoding human glyoxalase I into murine NIH3T3 cells. The recipient cells were shown to express elevated transcript and protein levels and a 10-fold increase in glyoxalase I enzyme activity. This was accompanied by an increased tolerance for exogenous methylglyoxal and enhanced resistance to the cytotoxic effects of two glyoxalase I inhibitors (s-p-bromobenzylglutathione diethyl ester and s-p-bromobenzylglutathione dicyclopentyl ester), a glutathione analogue [gamma-glutamyl-(S)-(benzyl)cysteinyl-(R)-(-)-phenylglycine diethyl ester] and the anti-cancer drugs mitomycin C and adriamycin. Steady-state levels of GSH were significantly lower in the transfected cells, perhaps reflecting increased flux as a consequence of elevated glyoxalase activity. This decrease did not alter the sensitivity to the alkylating agent chlorambucil. Although transfection did not affect the growth or doubling time of the NIH3T3 cells, analysis of glyoxalase I activity showed a consistent increase in tumour tissue when compared with pair-matched controls. Thus increased glyoxalase I is associated with the malignant phenotype and may also contribute to protection against the cytotoxicity of certain anti-cancer drugs. Images Figure 1 PMID:7619046

  9. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) promote adipogenesis in 3T3-L1 adipocyte cell culture.

    PubMed

    Kim, Jonggun; Sun, Quancai; Yue, Yiren; Yoon, Kyong Sup; Whang, Kwang-Youn; Marshall Clark, J; Park, Yeonhwa

    2016-07-01

    4,4'-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4'-dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 adipocytes. Treatment of DDT or DDE resulted in increased lipid accumulation accompanied by increased expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome-proliferator activated receptor-γ (PPARγ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase, and leptin. Moreover, treatment of DDT or DDE increased protein levels of C/EBPα, PPARγ, AMP-activated protein kinase-α (AMPKα), and ACC, while significant decrease of phosphorylated forms of AMPKα and ACC were observed. These finding suggest that increased lipid accumulation caused by DDT and DDE may mediate AMPKα pathway in 3T3-L1 adipocytes. PMID:27265825

  10. Isoflavonoids from Crotalaria albida Inhibit Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells via Suppression of PPAR-γ Pathway

    PubMed Central

    Sun, Qinhu; Chou, Guixin

    2015-01-01

    Two 2″-isopropenyl dihydrofuran isoflavonoids (1 and 3), one 2″-isopropenyl dihydrofuran chromone (2), as well as 13 known compounds were isolated from the herbs of Crotalaria albida. Their structures and relative configurations were elucidated via NMR and HRESIMS analyses. The 2″ S absolute configuration of 1 and 2 were deduced by comparing their NOESY spectra with that of 3, which was determined via single crystal X-ray diffraction (CuKα). The 3R absolute configuration of 1 was determined by CD. Compounds 1, 2, and 3 inhibit the adipocyte differentiation and lipid accumulation of 3T3-L1 through down-regulation of PPAR-γ activity. PMID:26285147

  11. Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells.

    PubMed

    Xu, Huiyun; Zhang, Jian; Wu, Jiawei; Guan, Ying; Weng, Yuanyuan; Shang, Peng

    2012-01-01

    Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin αvβ3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines. PMID:23096360

  12. A Small Molecule Swertisin from Enicostemma littorale Differentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice.

    PubMed

    Dadheech, Nidheesh; Soni, Sanket; Srivastava, Abhay; Dadheech, Sucheta; Gupta, Shivika; Gopurappilly, Renjitha; Bhonde, Ramesh R; Gupta, Sarita

    2013-01-01

    Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes. PMID:23662125

  13. A Small Molecule Swertisin from Enicostemma littorale Differentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice

    PubMed Central

    Dadheech, Nidheesh; Soni, Sanket; Srivastava, Abhay; Dadheech, Sucheta; Gupta, Shivika; Gopurappilly, Renjitha; Bhonde, Ramesh R.; Gupta, Sarita

    2013-01-01

    Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes. PMID:23662125

  14. AMP-activated protein kinase mediates insulin-like and lipo-mobilising effects of β-glucan-rich polysaccharides isolated from Pleurotus sajor-caju (Fr.), Singer mushroom, in 3T3-L1 cells.

    PubMed

    Kanagasabapathy, G; Chua, K H; Malek, S N A; Vikineswary, S; Kuppusamy, U R

    2014-02-15

    Mushrooms have been used to treat various diseases for thousands of years. In the present study, the effects of Pleurotus sajor-caju mushroom on lipogenesis, lipolysis and oxidative stress in 3T3-L1 cells were investigated. The β-glucan-rich polysaccharides (GE) from P. sajor-caju stimulated lipogenesis and lipolysis but attenuated protein carbonyl and lipid hydroperoxide levels in 3T3-L1 cells. This extract caused an increase in the expression of 5'-AMP-activated protein kinase subunit γ-2 (PKRAG2) and 5'-AMP-activated protein kinase subunit γ-3 (PKRAG3) when compared to control (untreated) cells. Moreover, GE induced the expressions of hormone-sensitive lipase, adipose triglyceride lipase enzymes, leptin, adiponectin and glucose transporter-4 in 3T3-L1 cells which may have contributed to the lipolytic and insulin-like activities observed in this study. These findings suggest that GE is a novel AMPK activator that may be valuable in the formulation of nutraceuticals and functional food for the prevention and treatment of diabetes mellitus. PMID:24128468

  15. RELAXIN enhances differentiation and matrix mineralization through Relaxin/insulin-like family peptide receptor 2 (Rxfp2) in MC3T3-E1 cells in vitro.

    PubMed

    Duarte, Carolina; Kobayashi, Yukiho; Kawamoto, Tatsuo; Moriyama, Keiji

    2014-08-01

    RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2. PMID:24857857

  16. Suppression of Adipogenesis by 5-Hydroxy-3,6,7,8,3',4'-Hexamethoxyflavone from Orange Peel in 3T3-L1 Cells.

    PubMed

    Wang, Yu; Lee, Pei-Sheng; Chen, Yi-Fen; Ho, Chi-Tang; Pan, Min-Hsiung

    2016-09-01

    We reported previously that hydroxylated polymethoxyflavones (HPMFs) effectively suppressed obesity in high-fat-induced mouse. In this study, we further investigated the molecular mechanism of action of 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF), one of major HPMFs in orange peel. Treatment of 5-OH-HxMF effectively inhibited lipid accumulation by 55-60% in a dose-dependent manner. The 5-OH-HxMF attenuated adipogenesis through downregulating adipogenesis-related transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs), as well as downstream target fatty acid synthase and acetyl-CoA carboxylase (ACC). 5-OH-HxMF activated adenosine monophosphate-activated protein kinase signaling and silent mating type information regulation 1 (SIRTUIN 1 or SIRT1) in 3T3-L1 adipocytes to decrease lipid accumulation. In addition, the inhibition rate of lipid accumulation was compared between 5-OH-HxMF and 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF). 5-OH-HxMF inhibited lipid accumulation 15-20% more than HpMF did, indicating that hydroxyl group at position 5 can be a key factor in the suppression of adipogenesis. PMID:27542074

  17. New vinegar produced by tomato suppresses adipocyte differentiation and fat accumulation in 3T3-L1 cells and obese rat model.

    PubMed

    Lee, Ju-Hye; Cho, Hyun-Dong; Jeong, Ji-Hye; Lee, Mi-Kyung; Jeong, Yong-Ki; Shim, Ki-Hwan; Seo, Kwon-Il

    2013-12-01

    There is an increasing surplus of tomatoes that are abandoned due to their failure to meet customer standards. Therefore, to allow both value additions and the effective reuse of surplus tomatoes, we developed tomato vinegar (TV) containing phytochemicals and evaluated its anti-obesity effects in vitro and in vivo. TV inhibited adipocyte differentiation of 3T3-L1 preadipocyte and lipid accumulation during differentiation. TV supplementation in rats fed a high-fat diet (HFD) markedly decreased visceral fat weights without changing the food and calories intakes. TV significantly decreased hepatic triglyceride and cholesterol levels compared to the HFD group. Furthermore, TV lowered plasma LDL-cholesterol level and antherogenic index compared to the HFD group, whereas elevated HDL-cholesterol to total cholesterol ratio. These results show that TV prevented obesity by suppressing visceral fat and lipid accumulation in adipocyte and obese rats, and suggest that TV can be used as an anti-obesity therapeutic agent or functional food. PMID:23871083

  18. Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type beta.

    PubMed Central

    Brown, K D; Holley, R W

    1987-01-01

    A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type beta (TGF-beta), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. A similar synergistic response has been demonstrated for TGF-beta purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-beta in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and we have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-beta and insulin converge at a post-receptor stage. PMID:3295869

  19. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    SciTech Connect

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  20. Effect of Nd:YAG laser-nitriding-treated titanium nitride surface over Ti6Al4V substrate on the activity of MC3T3-E1 cells.

    PubMed

    Wang, Min; Ning, Yingyuan; Zou, Haixiao; Chen, Si; Bai, Yi; Wang, Aihua; Xia, Haibin

    2014-01-01

    Ti6Al4V discs with a thickness of 2.5 mm and dimensions of 15 × 15 mm2 were fabricated. The titanium nitride (TiN) surface was formed via Nd:YAG laser-nitriding. A sandblast acid-etched (SA) surface was used as a control. Scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and surface roughness tests were conducted to study the surface and cross-section morphologies as well as the properties of TiN and SA surfaces. MC3T3-E1 osteoblast-like cells were cultured on the TiN and SA surfaces to evaluate the effect of TiN surface on cellular behaviors, including attachment, proliferation and differentiation. Morphological testing results revealed that the cross-section of TiN exhibited dendritic crystallization without cracking. The proliferation and differentiation of MC3T3-E1 cells on the laser-nitriding TiN surface were significantly increased compared to those cultured on SA surface. These findings suggested that the TiN surface generated from Nd:YAG laser-nitriding were favorable for the proliferation and differentiation of MC3T3-E1 cells, which is significant for implant surface modification. PMID:24211949

  1. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

    PubMed Central

    Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

    2016-01-01

    This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic

  2. A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin, Tested Over a Wide Range of Concentrations.

    PubMed

    Bonmati-Carrion, Maria Angeles; Alvarez-Sánchez, Nuria; Hardeland, Rüdiger; Madrid, Juan Antonio; Rol, Maria Angeles

    2013-01-01

    Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10-11-10-2 M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10-9-10-5 M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high-but not low-melatonin concentrations, although the mechanism of action still remains to be elucidated. PMID:23434670

  3. Transformation of BALB/c-3T3 cells: III. Development of a co-culture clonal survival assay for quantification of chemical cytotoxicity in high-density cell cultures.

    PubMed Central

    Matthews, E J

    1993-01-01

    A co-culture clonal survival assay was developed to measure the cytotoxicity of test chemical treatments to BALB/c-3T3 cells because the standard clonal survival assay using 200 wild type (WT) cells frequently overestimates chemical cytotoxicity when compared with identical treatment doses in high-density cultures. The assay used co-cultures of 3.2 x 10(4) WT cells, the same seeding density used in the transformation assay, and 200 ouabain resistant (OUAr) cells. After a 48-hr test chemical treatment, co-cultured cells were fed with culture medium containing 4 mM ouabain. The test chemical was cytotoxic to an equal percentage of WT and OUAr cells. Ouabain treatments killed the remaining WT cells. Thus, OUAr cells surviving the test chemical treatment measured the relative cloning efficiency (RCE) of all treated cells in the high-density cell co-culture. The co-culture assay succeeded because metabolic cooperation at the OUAr locus was not detected in BALB/c-3T3 cells. Five chemicals induced comparable cytotoxic responses in both assays, including actinomycin D, 5-bromo-2'-deoxyuridine, N'-methyl-N-nitro-N'-nitrosoguanidine, dimethyl sulfoxide and sodium chloride. In contrast, chemical cytotoxic responses detected in the standard and co-culture assays differed by > or = 10-fold for 11-aminoundecanoic acid, benzo[a]pyrene, cytosine arabinoside, and 3-methyl-cholanthrene and differed by > 2-fold for 2-acetylaminofluorene and dimethylnitrosamine. Detection of 11-aminoundecanoic acid-induced transformation was shown to be dependent on selecting treatment doses from the co-culture assay data. Thus, this method permits more accurate assessment of both chemical-induced cytotoxicity and transformation. PMID:8243400

  4. Transformation of BALB/c-3T3 cells: III. Development of a co-culture clonal survival assay for quantification of chemical cytotoxicity in high-density cell cultures.

    PubMed

    Matthews, E J

    1993-07-01

    A co-culture clonal survival assay was developed to measure the cytotoxicity of test chemical treatments to BALB/c-3T3 cells because the standard clonal survival assay using 200 wild type (WT) cells frequently overestimates chemical cytotoxicity when compared with identical treatment doses in high-density cultures. The assay used co-cultures of 3.2 x 10(4) WT cells, the same seeding density used in the transformation assay, and 200 ouabain resistant (OUAr) cells. After a 48-hr test chemical treatment, co-cultured cells were fed with culture medium containing 4 mM ouabain. The test chemical was cytotoxic to an equal percentage of WT and OUAr cells. Ouabain treatments killed the remaining WT cells. Thus, OUAr cells surviving the test chemical treatment measured the relative cloning efficiency (RCE) of all treated cells in the high-density cell co-culture. The co-culture assay succeeded because metabolic cooperation at the OUAr locus was not detected in BALB/c-3T3 cells. Five chemicals induced comparable cytotoxic responses in both assays, including actinomycin D, 5-bromo-2'-deoxyuridine, N'-methyl-N-nitro-N'-nitrosoguanidine, dimethyl sulfoxide and sodium chloride. In contrast, chemical cytotoxic responses detected in the standard and co-culture assays differed by > or = 10-fold for 11-aminoundecanoic acid, benzo[a]pyrene, cytosine arabinoside, and 3-methyl-cholanthrene and differed by > 2-fold for 2-acetylaminofluorene and dimethylnitrosamine. Detection of 11-aminoundecanoic acid-induced transformation was shown to be dependent on selecting treatment doses from the co-culture assay data. Thus, this method permits more accurate assessment of both chemical-induced cytotoxicity and transformation. PMID:8243400

  5. Single cell transfection using plasmid decorated AFM probes

    SciTech Connect

    Cuerrier, Charles M.; Lebel, Rejean; Grandbois, Michel . E-mail: michel.grandbois@usherbrooke.ca

    2007-04-13

    Eukaryotic cells were individually transfected using commercially available atomic force microscope tips decorated with plasmidic DNA encoding for the fluorescent protein EGFP. In a typical transfection attempt, the tip is forcibly incorporated into the cell thus allowing for the transfer of the genetic material through the cell membrane. A sharp discontinuity, corresponding to the passage of the tip through the cell membrane can be easily detected when monitoring the cellular deformation as a function of the applied force. In order for the transfection to be successful, the tip must reversibly penetrates the membrane without causing disturbance or damage to the cell. Transfection success rate (30%), cell survival, and growth are confirmed by epifluorescence microscopy. This technique provides an alternative tool to the transfection toolbox, allowing the transfection of specific individual cells with minimal disturbance.

  6. Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

    NASA Technical Reports Server (NTRS)

    Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

  7. Mitogen-stimulated events in nuclei of Swiss 3T3 cells. Evidence for a direct link between changes of inositol lipids, protein kinase C requirement and the onset of DNA synthesis.

    PubMed

    Martelli, A M; Gilmour, R S; Neri, L M; Manzoli, L; Corps, A N; Cocco, L

    1991-06-01

    Two different clones of Swiss 3T3 cells belonging to the same original cell line have been obtained, one of which was unresponsive to mitogenic stimulation (e.g. insulin-like growth factor-I, bombesin, insulin-like growth factor-I + bombesin), while the other clone showed a very high rate of DNA synthesis under identical conditions as demonstrated by 5-bromodeoxyuridine incorporation. Both types of cells expressed the IGF-I receptor and showed high contact inhibition. When highly purified nuclei from responsive cells, treated for a short time with bombesin and insulin-like growth factor-I or insulin-like growth factor-I alone, were incubated with [gamma-32P]adenosine triphosphate, the labelling of phosphatidylinositol-mono- and diphosphate decreased when compared to controls, while this transient effect did not appear in the nuclei from unresponsive cells. Similarly nuclear protein kinase C is activated only in responsive cells. Therefore, it seems that a direct link exists between polyphosphoinositide metabolism, protein kinase C activation and the early events leading to cell division, since the rapid changes in the labelling of both phosphatidylinositol mono- and di-phosphate occur only in nuclei from Swiss 3T3 cells, which respond to the mitogenic stimulus determined by insulin-like growth factor-I on its own, or in combination with bombesin. PMID:1646120

  8. Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.

    PubMed

    Lee, Joon Ho; Hur, Won

    2014-05-01

    This report describes fabrication of a millimeter-scale three-dimensional (3D) multicellular structure with a central cavity based on magnetic levitation of 3T3 cells that had ingested Fe3 O4 -containing microcapsules. Magnetically levitated cells initially formed a disc-shaped cell cluster at the air-medium interface and transformed into a spheroid (up to 2.8 mm in diameter) after 10-day incubation under a magnet. Hematoxylin-and-eosin-stained section revealed that an eosinophilic shell of cells enclosed a pale-staining core of the spheroid. Mitotic or elongated and aligned cells were found at the outer periphery of the shell, while Fe3 O4 deposits were distributed in the inner part of the shell. Surgical dissection indicated that the spheroid had a hollow interior filled with a fluid-state cell suspension. Accordingly, it was demonstrated that magnetically levitated 3T3 cells organized themselves into a tissue-like spheroid, resulting in core cell death. The spheroid can be used as a 3D tissue model and as building blocks that fused to form a more complicated structure. PMID:24254251

  9. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes

    NASA Astrophysics Data System (ADS)

    Torchilin, Vladimir P.; Levchenko, Tatyana S.; Rammohan, Ram; Volodina, Natalia; Papahadjopoulos-Sternberg, Brigitte; D'Souza, Gerard G. M.

    2003-02-01

    Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity (10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both in vitro and in vivo transfection with TATp-liposome-DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes in vitro. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome-DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment in vitro or ex vivo as well as for localized in vivo gene therapy.

  10. Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type. beta

    SciTech Connect

    Brown, K.D.; Holley, R.W.

    1987-06-01

    A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type ..beta.. (TGF-..beta..), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. /sup 125/I-EGF binding was measured and the efflux of /sup 45/Ca/sup 2 +/ was measured in response to mitogen stimulation. A similar synergistic response has been demonstrated for TGF-..beta.. purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-..beta.. in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and the authors have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-..beta.. and insulin converge at a post-receptor stage.

  11. Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

    SciTech Connect

    Minami, Yukiko . E-mail: ytakai@molbio.med.osaka-u.ac.jp

    2007-01-26

    Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceeds the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.

  12. Mechanism of the antiadipogenic-antiobesity effects of a rice hull smoke extract in 3T3-L1 preadipocyte cells and in mice on a high-fat diet.

    PubMed

    Kim, Sung Phil; Nam, Seok Hyun; Friedman, Mendel

    2015-09-01

    The present study investigated the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 preadipocyte cells and in mice fed high fat (HFD) and normal (ND) diets. At concentrations of 0.1% and 0.5%, RHSE was shown to reduce the cellular lipid content in MDI-induced 3T3-L1 cells by about 72% and 88%, respectively, compared to that in control cells without RHSE, indicating a strong antiadipogenic effect. This result was supported by the finding that the expression of the adipocyte differentiation marker adiponectin was suppressed. MTT and lactate dehydrogenase (LDH) release assays showed that RHSE doses of up to 0.5% (v/v) were not cytotoxic to the 3T3-L1 cells. RHSE activates AMP-activated protein kinase (AMPK) through raising the phosphorylated ratio during the early phase of cell differentiation, and western blot analysis showed that it dose-dependently inhibited the expression of the peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT-enhancer binding protein (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) at the late stage of differentiation. The antiadipogenic properties of RHSE were confirmed in vivo using experimental obese mice on a high-fat diet. Dietary supplementations of 0.5% and 1% RHSE resulted in a reduction at the end of the 7-week feeding study of body weight gain of 66.9% and 72.5%, respectively, a reduction of epididymal white adipose tissue weight by up to 87.9%, restoration of elevated total cholesterol and triglyceride levels in plasma and liver to those observed in the ND-fed mice, normalization of distorted serum leptin and adiponectin levels, and restoration of liver weight and glutamate oxaloacetate transaminase/glutamate pyruvate transaminase (GOT/GPT) enzymes, blood urea, and serum creatinine to the normal control levels observed in the ND-fed mice. As was found in the 3T3-L1 cells, RHSE up-regulated AMPK phosphorylation and down-regulated PPARγ, C/EBPα and SREBP-1c protein

  13. Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts

    PubMed Central

    Stockton, Rebecca A.; Jacobson, Bruce S.

    2001-01-01

    Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion

  14. miR-148a is a downstream effector of X-box-binding protein 1 that silences Wnt10b during adipogenesis of 3T3-L1 cells

    PubMed Central

    Cho, Yoon Mi; Kim, Tae-Min; Hun Kim, Dae; Hee Kim, Dong; Jeong, Seong-Whan; Kwon, Oh-Joo

    2016-01-01

    Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a. PMID:27055562

  15. Induction of interleukin 2 receptiveness and proliferation in resting peripheral T cells by monoclonal anti-CD3 (T3) antibodies does not require the presence of macrophages.

    PubMed Central

    Stingl, L A; Sinska, A; Landesmann, U; Smolen, J S

    1987-01-01

    In this study, we sought to elucidate the sequence of events by which mitogenic monoclonal anti-CD3 antibodies (anti-CD3-MoAb) initiate T cell activation. In cultures of monocyte-depleted resting T cells, two anti-CD3-MoAb, OKT3 and anti-Leu 4, induced a state of interleukin 2 (IL-2) receptiveness which culminated in T lymphocyte proliferation when recombinant IL-2 was provided. Evidence that Fc-receptor mediation by monocytes did not contribute to this mitogenesis was supported by studies showing that polyclonal F(ab')2 anti-mouse IgG Fc antibody did not alter the magnitude of the IL-2 driven T cell proliferative response, and by the use of T cells from donors whose monocytes were unable to assist in the induction of anti-Leu 4 (IgG1 subclass) initiated proliferation. Anti-CD3-MoAb, in the absence of IL-2, induced IL-2 receptor expression on purified T cells, and anti-IL 2 receptor antibodies inhibited T cell proliferation in the presence of this growth factor. Furthermore, following modulation of the CD3 molecular complex in the presence of monocytes, depletion of accessory cells rendered the modulated T cells mitogenically dependent on exogenous IL-2. IL-2 itself did not suffice to promote T cell proliferation in the absence of anti-CD3-MoAb. These results indicate that the binding of monoclonal antibody to CD3 is capable of initiating, in an accessory cell-independent manner, premitotic alterations in T cells which can culminate in proliferation when exogenous IL-2 is provided. PMID:3115639

  16. Pre-osteoblastic MC3T3-E1 promote breast cancer cell growth in bone in a murine xenograft model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cance...

  17. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  18. In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines

    PubMed Central

    Tomankova, Katerina; Polakova, Katerina; Pizova, Klara; Binder, Svatopluk; Havrdova, Marketa; Kolarova, Mary; Kriegova, Eva; Zapletalova, Jana; Malina, Lukas; Horakova, Jana; Malohlava, Jakub; Kolokithas-Ntoukas, Argiris; Bakandritsos, Aristides; Kolarova, Hana; Zboril, Radek

    2015-01-01

    One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg–DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg–DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg–DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg–DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression. PMID:25673990

  19. Boron nitride nanotubes chemically functionalized with glycol chitosan for gene transfection in eukaryotic cell lines.

    PubMed

    Ferreira, T H; Hollanda, L M; Lancellotti, M; de Sousa, E M B

    2015-06-01

    Nanostructured materials have been widely studied concerning their potential biomedical applications, primarily to selectively carry specific drugs or molecules within a tissue or organ. In this context, boron nitride nanotubes (BNNTs) have generated considerable interest in the scientific community because of their unique properties, presenting good chemical inertness and high thermal stability. Among the many applications proposed for BNNTs in the biomedical field in recent years, the most important include their use as biosensors, nanovectors for the delivery of proteins, drugs, and genes. In the present study, BNNTs were synthesized, purified, and functionalized with glycol chitosan through a chemical process, yielding the BNNT-GC. The size of BNNT-GC was reduced using an ultrasound probe. Two samples with different sizes were selected for in vitro assays. The nanostructures were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA), and dynamic light scattering (DLS). The in vitro assays MTT and neutral red (NR) were performed with NIH-3T3 and A549 cell lines and demonstrated that this material is not cytotoxic. Furthermore, the BNNT-GC was applied in gene transfection of plasmid pIRES containing a gene region that express a green fluorescent protein (GFP) in NIH-3T3 and A549 cell lines. The gene transfection was characterized by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Our results suggest that BNNT-GC has moderate stability and presents great potential as a gene carrier agent in nonviral-based therapy, with low cytotoxicity and good transfection efficiency. PMID:25231734

  20. Mammalian cell transfection: the present and the future

    PubMed Central

    Kim, Tae Kyung

    2010-01-01

    Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective. PMID:20549496

  1. Inhibition of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) by Gelidium elegans Using Alternative Drying and Extraction Conditions in 3T3-L1 and RAW 264.7 Cells

    PubMed Central

    Jeon, Hui-Jeon; Choi, Hyeon-Son; Lee, OK-Hwan; Jeon, You-Jin; Lee, Boo-Yong

    2012-01-01

    Gelidium (G.) elegans is a red alga inhabiting intertidal areas of North East Asia. We examined anti-oxidative and anti-inflammatory effects of G. elegans, depending on drying and extraction conditions, by determining reactive oxygen species (ROS) and nitric oxide (NO) in 3T3-L1 and RAW 264.7 cells. Extraction yields of samples using hot air drying (HD) and far-infrared ray drying (FID) were significantly higher than those using natural air drying (ND). The 70% ethanol extracts showed the highest total phenol and flavonoid contents compared to other extracts (0, 30, and 50% ethanol) under tested drying conditions. The scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitrite correlated with total phenol or flavonoid content in the extracts. The greatest DPPH scavenging effect was observed in 70% ethanol extract from FID and HD conditions. The production of ROS and NO in 3T3-L1 and macrophage cells greatly decreased with the 70% ethanol extraction derived from FID. This study suggests that 70% ethanol extraction of G. elegans dried by FID is the most optimal condition to obtain efficiently antioxidant compounds of G. elegans. PMID:24471073

  2. Anti-adipogenic activity of a new cyclic diarylheptanoid isolated from Alnus japonica on 3T3-L1 cells via modulation of PPARγ, C/EBPα and SREBP1c signaling.

    PubMed

    Sung, Sang Hyun; Lee, Mina

    2015-10-15

    Total methanolic extract of Alnus japonica fruits exhibited significant anti-adipogenic activities in 3T3-L1 cells. A new cyclic diarylheptanoid (1) along with ten known compounds (2-11) were isolated by activity-guided fractionation. Compound 1, determined to be 4-hydroxy-alnus-3,5-dione, showed the most potent anti-adipogenic effect. Compound 1 significantly down-regulated expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and sterol regulatory element binding protein 1 (SREBP1c) in 3T3-L1 cells, as determined by quantitative real-time PCR and Western blot analysis. Furthermore, compound 1 suppressed mRNA expression of C/EBPβ and C/EBPδ during the early stage of adipogenesis as well as stearoyl coenzyme A desaturase 1 (SCD-1) and fatty acid synthase (FAS), target genes of SREBP1c. Upon investigating the mechanism of natural products, we propose that cyclic diarylheptanoid (1), the most potent constituent of A. japonica, can be a potent therapeutic agent against obesity through anti-adipogenesis via down-regulation of PPARγ, C/EBPα, and SREBP1c signaling. PMID:26341132

  3. Correlation between cationic lipid-based transfection and cell division.

    PubMed

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. PMID:25556666

  4. Isolation of 1-monomethylphosphoinositol 4,5-bisphosphate (a product of methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate) from Swiss mouse 3T3 cells

    SciTech Connect

    Lips, D.L.; Bross, T.E.; Majerus, P.W.

    1988-01-01

    We have noted two previously undescribed inositol polyphosphates in neutral methanol extracts from Swiss mouse 3T3 cells that were grown in (/sup 3/H)inositol and stimulated with platelet-derived growth factor. They have been identified as 1-monomethylphosphoinositol 4,5-bisphosphate and 1-monomethylphosphoinositol 4-phosphate by comparison to a synthesized standard using HPLC chromatography, paper electrophoresis, and enzymatic dephosphorylation with inositol polyphosphate 5-phosphomonoesterase and intestinal alkaline phosphatase. We propose that these compounds are formed by methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate and inositol 1,2-(cyclic)-4-bisphosphate present in the cells. Inositol cyclic phosphates did not react with neutral methanol in the absence of the cells, which are required for the methanolysis reaction. These findings suggest a role for inositol cyclic phosphates as reactive compounds that are added to as yet unidentified cellular acceptors.

  5. PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway.

    PubMed

    Li, Haiying; Cui, Yazhou; Luan, Jing; Zhang, Xiumei; Li, Chengzhi; Zhou, Xiaoyan; Shi, Liang; Wang, Huaxin; Han, Jinxiang

    2016-02-12

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation. PMID:26809092

  6. Mammalian target of rapamycin complex 1 (mTORC1) plays a role in Pasteurella multocida toxin (PMT)-induced protein synthesis and proliferation in Swiss 3T3 cells.

    PubMed

    Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A; Chock, P Boon

    2013-01-25

    Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the α subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in Gα(q/11) knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of Gα(q/11), which leads to the activation of PLCβ to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gα(q/11)/PLCβ/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway. PMID:23223576

  7. Identification of cytoprotective constituents of the flower buds of Tussilago farfara against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells.

    PubMed

    Kang, Unwoo; Park, Jiyoung; Han, Ah-Reum; Woo, Mi Hee; Lee, Je-Hyun; Lee, Sang Kook; Chang, Tong-Shin; Woo, Hyun Ae; Seo, Eun Kyoung

    2016-04-01

    A new cytoprotective compound, 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone (1) was isolated from the flower buds of Tussilago farfara L. (Compositae), together with eight known compounds, 3,4-dicaffeoyl isoquinic acid (2), trans-cinnamic acid (3), 4-hydroxyacetophenone (4), 4,5-dicaffeoylquinic acid methyl ester (5), 3,5-dicaffeoylquinic acid methyl ester (6), 4-hydroxybenzoic acid (7), isoquercetrin (8), and ligucyperonol (9). Compounds 2-4 were found in this plant for the first time. The isolates 1-9, were tested for their cytoprotective activities against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells. Among them, 1 and 3 showed significant cytoprotective activities as determined by MTT assay and lactate dehydrogenase leakage, indicating their possibility as the potent cytoprotective agents. The structure of 1 was determined by spectroscopic data analysis including 1D- and 2D-NMR experiments, and its absolute configuration was elucidated by a circular dichroism. PMID:26983826

  8. Electroporation for Transfection and Differentiation of Dental Pulp Stem Cells

    PubMed Central

    Rabie, Bakr M.

    2013-01-01

    Abstract Target gene delivery is needed to induce cellular differentiation or a specific therapeutic effect. Electroporation is a relatively safe and simple technique to deliver nucleic acids to the cell that acts by rendering cells transiently permeable using short periods of high voltage. In stem cell research, human dental pulp stem cells (hDPSCS) are highly accessible, and they exhibit broad differentiation potential. Until now, no studies have attempted to optimize electroporation parameters for DPSCs with respect to transfection efficiency and viability. In this study, we aimed to optimize transfection of DPSCs through varying different electroporation parameters, including voltage, mode of pulsation, and the number of pulses. As positive control, we used commonly utilized the chemical transfection reagents Lipofectamine 2000 and FuGene 6. In addition, we used our newly optimized transfection conditions to transfect hDPSCs with a functional chondrogenic transgene. We obtained higher transfection efficiency and cell viability with these electroporation conditions compared to controls. The highest transfection efficiency (63.81±4.72%) was achieved with 100 V, 20 msec, one-pulse square-wave condition. Among chemical transfection groups, FuGene 6 showed the highest cell viability at all tested transfection ratios, while Lipofectamine 2000 showed the highest transfection efficiency (19.23±3.19%) using 1:1 DNA (μg):Lipofectamine (μL). Transfected DPSCs functionally expressed the transforming growth factor β-3 chondrogenic transgene on the mRNA level as detected by real-time polymerase chain reaction and on the protein level as detected by Western blot analysis. An increase in various chondrogenic markers was also found when studying mRNA expression in transfected cells. In conclusion, the results of our study demonstrate optimal electroporation and chemical transfection reagent conditions for hDPSCs, and, subsequently, we provide proof of concept for

  9. Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

    PubMed Central

    Rettenmier, C W; Roussel, M F; Ashmun, R A; Ralph, P; Price, K; Sherr, C J

    1987-01-01

    NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions. Images PMID:3039346

  10. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. PMID:26529667

  11. MC3T3-E1 Cell Response to Ti1-xAgx and Ag-TiNx Electrodes Deposited on Piezoelectric Poly(vinylidene fluoride) Substrates for Sensor Applications.

    PubMed

    Marques, S M; Rico, P; Carvalho, I; Gómez Ribelles, J L; Fialho, L; Lanceros-Méndez, S; Henriques, M; Carvalho, S

    2016-02-17

    In the sensors field, titanium based coatings are being used for the acquisition/application of electrical signals from/to piezoelectric materials. In this particular case, sensors are used to detect dynamic mechanical loads at early stages after intervention of problems associated with prostheses implantation. The aim of this work is to select an adequate electrode for sensor applications capable, in an initial stage to avoid bone cell adhesion, but at a long stage, permit osteointegration and osteoinduction. This work reports on the evaluation of osteoblast MC3T3-E1 cells behavior in terms of proliferation, adhesion and long-term differentiation of two different systems used as sensor electrodes: Ti1-xAgx and Ag-TiNx deposited by d.c. and pulsed magnetron sputtering at room temperature on poly(vinylidene fluoride) (PVDF). The results indicated an improved effect of Ag-TiNx electrodes compared with Ti1-xAgx and TiN, in terms of diminished cell adhesion and proliferation at an initial cell culture stage. Nevertheless, when cell culture time is longer, cells grown onto Ag-TiNx electrodes are capable to proliferate and also differentiate at proper rates, indicating the suitability of this coating for sensor application in prostheses devices. Thus, the Ag-TiNx system was considered the most promising electrode for tissue engineering applications in the design of sensors for prostheses to detect dynamic mechanical loads. PMID:26840928

  12. N-acetyl cysteine inhibits H2O2-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway.

    PubMed

    Lee, Daewoo; Kook, Sung-Ho; Ji, Hyeok; Lee, Seung-Ah; Choi, Ki-Choon; Lee, Kyung-Yeol; Lee, Jeong-Chae

    2015-11-01

    There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H2O2), N-acetyl cysteine (NAC), or both. Exposing the cells to H2O2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H2O2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H2O2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H2O2 to near normal levels in the cells. Collectively, our findings suggest that H2O2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC. PMID:26303969

  13. In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems.

    PubMed

    Sauer, Ursula G; Vogel, Sandra; Hess, Annemarie; Kolle, Susanne N; Ma-Hock, Lan; van Ravenzwaay, Bennard; Landsiedel, Robert

    2013-02-01

    The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured. PMID:23085368

  14. Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells.

    PubMed Central

    Chepelinsky, A B; Seif, R; Martin, R G

    1980-01-01

    We studied the pattern of integration of the simian virus 40 (SV40) genome into the cellular DNA of N-transformants (temperature sensitive) and A-transformants (temperature insensitive) derived from 3T3-Fisher rat and Chinese hamster lung cells. The SV40 DNA was covalently linked to the cellular DNA in both types of transformants. In the rat cells, most N-transformants contained SV40 sequences integrated at a single site; most A-transformants contained SV40 sequences integrated at two to five sites. In the Chinese hamster cells, no significant correlation between the number of integration sites and the phenotype of the transformant was found; one of three integration sites were observed for both the N- and A-transformants. Single copies and tandem repeats of SV40 sequences were observed in A- and N-transformants derived from rat cells. A-transformants arise neither by amplification of the SV40 genome nor by integration at a unique site. Images PMID:6251267

  15. Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.

    PubMed

    Lee, S; Shin, S; Jung, E; Park, D

    2016-08-01

    The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity. PMID:26281901

  16. Curcuma longa polyphenols improve insulin-mediated lipid accumulation and attenuate proinflammatory response of 3T3-L1 adipose cells during oxidative stress through regulation of key adipokines and antioxidant enzymes.

    PubMed

    Septembre-Malaterre, Axelle; Le Sage, Fanny; Hatia, Sarah; Catan, Aurélie; Janci, Laurent; Gonthier, Marie-Paule

    2016-07-01

    Plant polyphenols may exert beneficial action against obesity-related oxidative stress and inflammation which promote insulin resistance. This study evaluated the effect of polyphenols extracted from French Curcuma longa on 3T3-L1 adipose cells exposed to H2 O2 -mediated oxidative stress. We found that Curcuma longa extract exhibited high amounts of curcuminoids identified as curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which exerted free radical-scavenging activities. Curcuma longa polyphenols improved insulin-mediated lipid accumulation and upregulated peroxisome proliferator-activated receptor-gamma gene expression and adiponectin secretion which decreased in H2 O2 -treated cells. Curcuminoids attenuated H2 O2 -enhanced production of pro-inflammatory molecules such as interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nuclear factor κappa B. Moreover, they reduced intracellular levels of reactive oxygen species elevated by H2 O2 and modulated the expression of genes encoding superoxide dismutase and catalase antioxidant enzymes. Collectively, these findings highlight that Curcuma longa polyphenols protect adipose cells against oxidative stress and may improve obesity-related metabolic disorders. © 2016 BioFactors, 42(4):418-430, 2016. PMID:27094023

  17. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  18. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    PubMed

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. PMID:25957917

  19. Transient transfection of mammalian cells using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  20. Multiplexing nano-electroporation for simultaneous transfection of multiple cells

    NASA Astrophysics Data System (ADS)

    Howdyshell, M.; Vieira, G.; Gallego-Perez, D.; Zhao, X.; Lee, L. J.; Sooryakumar, R.

    2013-03-01

    Transfection of biomolecules into cells via electrophoresis across nanochannels, or nano-electroporation, is a recently developed technique shown to deliver precisely controlled dosages with low cell mortality rates. Such advantages are due to the nanochannels used for transfection, which distinguish this technique from bulk and micro-electroporation. Recent demonstrations of nano-electroporation rely on optical tweezers for cell localization, which restrict throughput to sequential electroporation of one cell at a time. In the current work, we overcome this drawback by advancing a multiplexed approach that integrates the nano-channel device with an array of magnetic traps remotely controlled by external magnetic fields. This setup enables multiple magnetically labeled cells to be manipulated in parallel, allowing for simultaneous electroporation of many cells with precisely controlled dosages. After transfection, the cells can be moved downstream for further analysis. Such a magnetically-actuated, remotely-controlled approach for loading of cells and subsequent removal of transfected cells has the potential to transform the current device into an automated platform for simultaneous dosage-controlled biomolecule delivery to large numbers of individual cells.

  1. Comparison of alkaline phosphatase activity of MC3T3-E1 cells cultured on different Ti surfaces: modified sandblasted with large grit and acid-etched (MSLA), laser-treated, and laser and acid-treated Ti surfaces

    PubMed Central

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P<.05). Furthermore, ALP expression levels of MSLA and LAT surfaces were significantly higher than expression levels of LT surface-adherent cells at 7, 14, and 21 days, respectively (P<.05). However, ALP expression levels between MSLA and LAT surface were equal at 7, 14, and 21 days (P>.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

  2. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  3. Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Promote Adipocyte Differentiation and Inhibit Inflammation in 3T3-F442A Cells

    PubMed Central

    Chakrabarti, Subhadeep; Wu, Jianping

    2015-01-01

    Milk derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE). Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB) pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease. PMID:25714093

  4. Wnt/β-catenin signaling plays an important role in the protective effects of FDP-Sr against oxidative stress induced apoptosis in MC3T3-E1 cell.

    PubMed

    Qi, Huan-Huan; Bao, Jun; Zhang, Qi; Ma, Bo; Gu, Gui-Ying; Zhang, Peng-Ling; Ou-Yang, Gang; Wu, Zi-Mei; Ying, Han-Jie; Ou-Yang, Ping-Kai

    2016-10-01

    Strontium fructose 1,6-diphosphate (FDP-Sr) is a new strontium-containing compound. The primary aim of this study was to clarify whether the structure component of FDP-Sr, FDP could benefit the protective effect of Sr (II) against oxidative stress induced apoptosis, and meanwhile to further explore the important role of Wnt/β-catenin signaling in the anti-apoptosis effect of FDP-Sr in response to oxidative stress induced by H2O2 in an osteoblastic MC3T3-E1 cell line. Results showed that FDP-Sr could improve the osteoblastic differentiation under oxidative stress with induced cell proliferation and improved mineralization. The inhibition effect of FDP-Sr on cell apoptosis induced by H2O2 was proved by reduced reactive oxygen species production and activated caspase3. Under oxidative stress, mRNA and protein levels of phospho-β-catenin reduced, while β-catenin increased in the FDP-Sr treatment cell, leaded to the up-regulations of Runx2 and OPG at both mRNA and protein levels, finally improved the differentiation of osteoblasts. By the engagement of Wnt/β-catenin pathway's inhibitor (XAV-939), the protective effects of FDP-Sr on osteoblastic differentiation against oxidative stress were repressed along with inhibited wnt/β-catenin signaling and reduced mRNA and protein levels of Runx2 and OPG. In conclusion, FDP-Sr was demonstrated to protect osteoblast differentiation from oxidative damage induced by H2O2 through up-regulation of Wnt/β-catenin signaling, and FDP in FDP-Sr was able to directly improve the oxidative stress injury through its ROS scavenging ability. PMID:27575480

  5. Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA

    SciTech Connect

    Schneider, C.A.; Lim, R.W.; Terwilliger, E.; Herschman, H.R.

    1986-01-01

    The authors have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind /sup 125/I-labeled EGF; each lacks a functional EGF receptor. They used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in /sup 125/I-labeled cell extracts or in (/sup 35/S)methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

  6. Photoporation and cell transfection using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  7. STAT5a promotes the transcription of mature mmu-miR-135a in 3T3-L1 cells by binding to both miR-135a-1 and miR-135a-2 promoter elements.

    PubMed

    Wei, Xiajie; Cheng, Xiaoyan; Peng, Yongdong; Zheng, Rong; Chai, Jin; Jiang, Siwen

    2016-08-01

    Despite extensive research on the role of miR-135a in biological processes, very little attention has been paid to the regulation of its transcription. We have previously reported that miR-135a suppresses 3T3-L1 preadipocyte differentiation and adipogenesis by directly targeting the adenomatous polyposis coli (APC) gene and activating the canonical Wnt/β-catenin signaling pathway, but the regulatory elements that regulate the expression of the two isoforms of miR-135a (miR-135a-1 and miR-135a-2) remain poorly understood. Here, by using deletion analysis, we predicted two binding sites (-874/-856 and -2020/-2002) for the transcription factor Signal Transducers and Activators of Transcription 5a (STAT5a) within the core promoters of miR-135a-1 and miR-135a-2 (-1128/-556 and -2264/-1773), and the subsequent site-directed mutagenesis indicated that the two STAT5a binding sites regulated the activity of the miR-135a-1 and miR-135a-2 promoters. The binding of STAT5a to the miR-135a-1/2 core promoters in vitro and in cell culture was identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays. Overexpression and RNAi knockdown of STAT5a showed that the transcription factor regulated the endogenous miR-135a expression. Additionally, The expression time frame of STAT5a and APC indicated a potential negative feedback between them. In sum, the overall results from this study indicate that STAT5a regulates miR-135a transcription by binding to both miR-135a-1 and miR135a-2 promoter elements and the findings provide novel insights into the molecular regulatory mechanisms of miR-135a during adipogenesis. PMID:27276245

  8. A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor.

    PubMed

    Quarles, L D; Hartle, J E; Siddhanti, S R; Guo, R; Hinson, T K

    1997-03-01

    The presence of a cation-sensing mechanism in osteoblasts is suggested by the ability of specific cations to stimulate osteoblastic proliferation in culture and to induce de novo bone formation in some experimental models. Our study examines whether extracellular cations stimulate osteoblasts through the recently identified G protein-coupled calcium receptor (CaR). We found that CaR agonists, calcium (Ca2+), gadolinium (Gd3+), aluminum (Al3+), and neomycin, stimulated DNA synthesis in murine-derived MC3T3-E1 preosteoblasts, whereas magnesium (Mg2+), nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+) had no effect. With the exception of Mg2+, the cation specificities and apparent affinities were similar to that reported for CaR. CaR agonists also stimulated DNA synthesis in C3HT10(1/2) fibroblasts, but not in mesangial PVG, CHO, hepatic HTC, COS-7 cells, or malignant transformed ROS17/2.8 and UMR-106 osteoblasts. In addition, similar to other growth factors, CaR agonists activated transcription of a serum response element luciferase reporter construct (SRE-Luc) stably transfected into MC3T3-E1 osteoblasts, but had no effect on SRE-Luc transfected into CHO and COS-7 cells. We were unable to detect CaR expression by Northern analysis using a mouse CaR-specific probe or to amplify CaR mRNA by reverse transcribed polymerase chain reaction in MC3T3-E1 osteoblasts. These findings suggest that an extra-cellular cation-sensing mechanism is present in murine-derived osteoblasts that is functionally similar to but molecularly distinct from CaR. PMID:9076582

  9. Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes.

    PubMed Central

    Brown, N F; Hill, J K; Esser, V; Kirkland, J L; Corkey, B E; Foster, D W; McGarry, J D

    1997-01-01

    The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the beta-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of tis choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used

  10. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  11. Efficient Gene Transfection into Mammalian Cells Mediated by Cross-linked Polyethylenimine

    PubMed Central

    Dong, Wei; Li, Shufeng; Jin, Guanghui; Sun, Qiming; Ma, Dingyuan; Hua, Zichun

    2007-01-01

    25 kDa branched polyethylenimine (PEI) has successfully been used for in vitro and in vivo gene delivery approaches, but it is cytotoxic. Smaller PEIs are usually non-cytotoxic but less efficient. In order to enhance the gene delivery efficiency and minimize cytotoxicity of PEI, we explored to synthesize cross-linked PEIs with degradable bonds by reacting amines of small branched 2000 Da PEI with small diacrylate (1,4-butanediol diacrylate or ethyleneglycol dimethacrylate) for 2–6 hours. The efficiency of the cross-linked PEIs during in vitro delivering plasmid containing enhanced green fluorescent protein (EGFP) gene reporter and their cytotoxicity were assessed in melanoma B16F10 cell and other cell lines. In vivo gene delivery efficiency was evaluated by direct injection delivery of the EGFP plasmid/cross-linked PEI complexes into mice and by estimating the EGFP expression in animal muscles. Compared to commercially available 25-kDa branched PEI, the cross-linked PEIs reported here could mediate more efficient expression of reporter gene than the 25-kDa PEI control, 19-fold more efficiently in B16F10 cells, 17-fold in 293T cells, 2.3-fold in 3T3 cells, and they exhibited essentially nontoxic at their optimized condition for gene delivery. Furthermore the transfection activity of polyplexs was preserved in the presence of serum proteins. The muscle transfected with the cross-linked PEI prepared here exhibited normal morphology and excellent gene expression. The cross-linked PEIs reported here were evidently more efficient than the commercial 25-kD PEI control and had less cytotoxicity in gene delivery in vitro and in vivo.

  12. Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes

    SciTech Connect

    Bick, T.; Frick, G.P.; Leonard, D.

    1994-12-31

    In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHR{sub L}) and a smaller isoform, GHR{sub S} that consists of the extracellular domain and a unique hydrophillic carboxyl terminus. We examined the hypothesis that GHR{sub S} may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHR{sub S} was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHR{sub s} in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHR{sub L} in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHR{sub L}. The transfected cDNA for GHR{sub S} directs the synthesis of a 50 kDa protein. We conclude that GHR{sub S} contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHR{sub S} in 3T3-L1 cells altered cell function in the absence of GH. 20 refs., 4 figs.

  13. Application of the improved BALB/c 3T3 cell transformation assay to the examination of the initiating and promoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic carcinogen study group of Japan.

    PubMed

    Tsuchiya, Toshiyuki; Umeda, Makoto; Tanaka, Noriho; Sakai, Ayako; Nishiyama, Hiroshi; Yoshimura, Isao; Ajimi, Syozo; Asada, Shin; Asakura, Masumi; Baba, Hiroshi; Dewa, Yasuaki; Ebe, Youji; Fushiwaki, Yuichi; Hagiwara, Yuji; Hamada, Shuichi; Hamamura, Tetsuo; Iwase, Yumiko; Kajiwara, Yoshitsugu; Kasahara, Yasushi; Kato, Yukihiko; Kawabata, Masayoshi; Kitada, Emiko; Kaneko, Kazuko; Kizaki, Yuko; Kubo, Kinya; Miura, Daisaku; Mashiko, Kaori; Mizuhashi, Fukutaro; Muramatsu, Dai; Nakajima, Madoka; Nakamura, Tetsu; Oishi, Hidetoshi; Sasaki, Toshiaki; Shimada, Sawako; Takahashi, Chitose; Takeda, Yuko; Wakuri, Sinobu; Yajima, Nobuhiro; Yajima, Satoshi; Yatsushiro, Tomoko

    2010-03-01

    The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1microg/ml 12-O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the twostage assay (pretreated with 0.2microg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-beta1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative - again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the

  14. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity.

    PubMed

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography-Mass Spectrometry (GC-MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  15. Effect of copper-doped silicate 13-93 bioactive glass scaffolds on the response of MC3T3-E1 cells in vitro and on bone regeneration and angiogenesis in rat calvarial defects in vivo.

    PubMed

    Lin, Yinan; Xiao, Wei; Bal, B Sonny; Rahaman, Mohamed N

    2016-10-01

    The release of inorganic ions from biomaterials could provide an alternative approach to the use of growth factors for improving tissue healing. In the present study, the release of copper (Cu) ions from bioactive silicate (13-93) glass scaffolds on the response of cells in vitro and on bone regeneration and angiogenesis in vivo was studied. Scaffolds doped with varying concentrations of Cu (0-2.0wt.% CuO) were created with a grid-like microstructure by robotic deposition. When immersed in simulated body fluid in vitro, the Cu-doped scaffolds released Cu ions into the medium in a dose-dependent manner and converted partially to hydroxyapatite. The proliferation and alkaline phosphatase activity of pre-osteoblastic MC3T3-E1 cells cultured on the scaffolds were not affected by 0.4 and 0.8wt.% CuO in the glass but they were significantly reduced by 2.0wt.% CuO. The percent new bone that infiltrated the scaffolds implanted for 6weeks in rat calvarial defects (46±8%) was not significantly affected by 0.4 or 0.8wt.% CuO in the glass whereas it was significantly inhibited (0.8±0.7%) in the scaffolds doped with 2.0wt.% CuO. The area of new blood vessels in the fibrous tissue that infiltrated the scaffolds increased with CuO content of the glass and was significantly higher for the scaffolds doped with 2.0wt.% CuO. Loading the scaffolds with bone morphogenetic protein-2 (1μg/defect) significantly enhanced bone infiltration and reduced fibrous tissue in the scaffolds. These results showed that doping the 13-93 glass scaffolds with up to 0.8wt.% CuO did not affect their biocompatibility whereas 2.0wt.% CuO was toxic to cells and detrimental to bone regeneration. PMID:27287141

  16. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    PubMed Central

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  17. Application of nanostructured biochips for efficient cell transfection microarrays

    NASA Astrophysics Data System (ADS)

    Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  18. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  19. Laser-based patterning for transfected cell microarrays.

    PubMed

    Hook, Andrew L; Creasey, Rhiannon; Hayes, Jason P; Thissen, Helmut; Voelcker, Nicolas H

    2009-12-01

    The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics. PMID:20811112

  20. In vitro transformation of mouse testis cells by oncogene transfection.

    PubMed

    Morimoto, Hiroko; Lee, Jiyoung; Tanaka, Takashi; Ishii, Kei; Toyokuni, Shinya; Kanatsu-Shinohara, Mito; Shinohara, Takashi

    2012-05-01

    Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming. PMID:22357549

  1. Cell transfection as a tool to study growth hormone action

    SciTech Connect

    Norstedt, G.; Enberg, B.; Francis, S.

    1994-12-31

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determined pathways whereby GH signals are conveyed within the cell. 33 refs., 2 tabs.

  2. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    PubMed

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

  3. Chito-Oligosaccharide Inhibits the De-Methylation of a ‘CpG’ Island within the Leptin (LEP) Promoter during Adipogenesis of 3T3-L1 Cells

    PubMed Central

    Bahar, Bojlul; O’Doherty, John V.; O’Doherty, Alan M.; Sweeney, Torres

    2013-01-01

    Chito-oligosaccharide (COS) is a natural bioactive compound, which has been shown to suppress lipid metabolic genes and lipid accumulation in differentiating adipocytes. Leptin has been identified as a key regulator of energy homeostasis and is known to be under epigenetic regulation during adipogenesis. Hence, the first objective of this experiment was to compare leptin gene (LEP) expression and leptin secretion during the different stages of adipogenesis and to investigate the effect of COS on these processes. As COS inhibited LEP expression during adipogenesis, the second aim was to investigate the methylation dynamics of a ‘CpG’ island in the proximal region of the LEP promoter during adipogenesis and to determine the effect of COS on this process. Mouse 3T3-L1 cells were stimulated to differentiate in the absence or presence of COS and the levels of leptin mRNA and protein were evaluated on days 0, 2, 4 and 6 post-induction of differentiation (PID). The extent of de-methylation of six CpG sites was evaluated. LEP mRNA transcript and protein could not be detected on either day 0PID or 2PID. In contrast, both were detected on day 4PID (P<0.05) and 6PID (P<0.001) and both were inhibited by COS (P<0.001). Of the six CpG sites analyzed, CpG_52, CpG_62 and CpG_95 became 11.5, 5.0 and 5.0% de-methylated between day 2PID and 6PID, respectively. COS blocked this de-methylation event at CpG_52 (P<0.001), CpG_62 (P<0.01) and CpG_95 (P<0.01) on day 6PID. These data suggest that COS can have an epigenetic effect on differentiating adipocytes, a novel biological function of COS which has potential applications for the manipulation of leptin gene expression, adipogenesis, and conditions within the metabolic syndrome spectrum. PMID:23544120

  4. Paradoxical regulation of dopamine receptors in transfected 293 cells.

    PubMed

    Filtz, T M; Artymyshyn, R P; Guan, W; Molinoff, P B

    1993-08-01

    Selective expression of subtypes of receptors in mammalian cell lines permits the study of the regulation of receptors in a homogeneous population of cells growing under controlled conditions. cDNAs encoding the human D2L and D2S receptors were ligated into a eukaryotic expression vector, pRc/CMV. The resulting plasmid, which contains a cytomegalovirus promoter for high expression levels, was used for stable transfection of 293 cells, a human kidney cell line. Expression of D2L and D2S receptors in 293 cells was confirmed by radioligand binding assays with [125I]NCQ 298. The pharmacological properties of the expressed receptors were comparable to those of receptors in rat striatal homogenates and in other transfected cell lines. D2L and D2S receptors were coupled to inhibition of cAMP accumulation in 293 cells. Incubation of 293-D2L cells with agonists resulted in an increase in the density of D2 receptors without a change in the affinity of the receptors for [125I]NCQ 298. This effect was time dependent, with a t1/2 of approximately 6 hr. The dose dependence of up-regulation followed the pharmacological profile expected of a D2 receptor, with an order of potency of N-propylnorapomorphine (NPA) > quinpirole > dopamine. The density of receptors was further increased by incubation of cells with agonist together with forskolin or 8-bromo-cAMP. D2S receptors responded similarly to D2L receptors to treatment with NPA and forskolin. Exposure of 293-D2L cells to the beta-adrenergic receptor agonist isoproterenol did not change the density of D2L receptors. Similarly, NPA had no effect on levels of endogenously expressed beta-adrenergic receptors in 293-D2L cells, as assayed by binding of [125I]iodocyanopindolol. Levels of beta-adrenergic receptors in transfected 293-beta 2 or 293-D2L cells did not increase after exposure to NPA but decreased after exposure to isoproterenol. Cells expressing D2L receptors were incubated with antagonists, including SCH-23390, sulpiride

  5. Immune Monitoring Using mRNA-Transfected Dendritic Cells.

    PubMed

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by mRNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA. PMID:27236804

  6. The effect of the suspension cells in plasma gene transfection method

    NASA Astrophysics Data System (ADS)

    Isozaki, Yuki; Nakano, Koki; Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Tachibana, Kunihide; Jinno, Masafumi

    2015-09-01

    Plasma gene transfection method is a unique technique for introducing nucleic acids into cells by using plasma irradiation. In our previous works, plasma gene transfection method was performed for the adherent cells, e.g. COS-7 cells, and the influence of plasma on gene transfection has been investigated. As a next step for plasma medicine, transfection to much more various kinds of target cells is required. In this study, the authors attempted gene transfection to two kinds of suspension and four kinds of adherent cells. Although the transfection ratios to the suspension cells were low, transfection to all the kinds of cells were validated. To upregulate the transfection ratio for suspension cells, the authors are validating related factors by plasma irradiation. This work was partly supported by JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (Number 25108509, 15H00896) and a grant from Ehime University.

  7. Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.

    PubMed Central

    Reed, B C; Kaufmann, S H; Mackall, J C; Student, A K; Lane, M D

    1977-01-01

    Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant. PMID:303773

  8. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

    Background Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. Results Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). Conclusions These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle. PMID:23521774

  9. Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2015-01-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e. better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials. PMID:24749393

  10. Software-aided automatic laser optoporation and transfection of cells

    NASA Astrophysics Data System (ADS)

    Georg Breunig, Hans; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2015-06-01

    Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.

  11. Altered cholesterol metabolism in APP695-transfected neuroblastoma cells.

    PubMed

    Wirths, Oliver; Thelen, Karin M; Lütjohann, Dieter; Falkai, Peter; Bayer, Thomas A

    2007-06-01

    Cholesterol has been implicated to play an important role in the generation of Abeta peptides, which are the main component of beta-amyloid plaques in the brains of patients suffering from Alzheimer's disease (AD). Epidemiological data implicate that lowering cholesterol levels has beneficial effects on the extent of beta-amyloid pathology. Thus therapeutic intervention using cholesterol lowering drugs like statins seems to be a promising approach. A couple of studies, in vitro or in vivo by the use of AD transgenic mouse models, focused on the manipulation of cholesterol levels and the resulting effects on Abeta generation. In contrast, there is not much known about the effect of the amyloid precursor protein (APP) on cholesterol levels. In the present report, we transfected human neuroblastoma cells with human APP695 and compared cellular cholesterol levels with the respective levels in Mock-transfected control cells. Furthermore, we determined the levels of diverse cholesterol precursors and metabolites using gas chromatography-mass spectrometry (GC-MS). Significant differences in the levels of the respective cholesterol precursors were observed, whereas inhibition of gamma-secretase activity by the gamma-secretase inhibitor DAPT did not have a significant effect on cellular cholesterol metabolism. PMID:17428449

  12. Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems.

    PubMed

    Selmeczi, David; Hansen, Thomas Steen; Met, Özcan; Svane, Inge Marie; Larsen, Niels B

    2016-01-01

    Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75 % with survival rates of >90 %. This chapter describes the instrumentation and methods needed for the efficient transfection by electroporation of millions of dendritic cells in one continuous flow process. PMID:27236798

  13. Dentin barrier test with transfected bovine pulp-derived cells.

    PubMed

    Schmalz, G; Schuster, U; Thonemann, B; Barth, M; Esterbauer, S

    2001-02-01

    Growth kinetics of SV40 large T-antigen-transfected bovine pulp-derived cells on dentin were investigated. These cells were used in a dentin barrier test device, and the system was evaluated by testing a set of dental filling materials. Cells (120 cells/mm2) were seeded on dentin slices and incubated for up to 21 days. Cell proliferation was recorded using MTT assay. For cytotoxicity tests 3500 cells/mm2 were seeded on dentin discs, which were then incorporated into the dentin barrier test device. After 72 h preincubation test materials were applied. After a 24 h exposure with or without perfusion of the pulpal part of the test device, cell survival was evaluated using MTT assay. The cells revealed similar growth kinetics on dentin slices and on tissue culture plates. In cytotoxicity tests the cells were more sensitive toward the test materials than previously used three-dimensional cultures of human foreskin fibroblasts and as anticipated from clinical experience. Further improvement is expected by using three-dimensional cultures of pulp-derived cells. PMID:11491647

  14. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    PubMed

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation. PMID:25887802

  15. DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    PubMed Central

    Kuroda, Masashi; Tominaga, Ayako; Nakagawa, Kasumi; Nishiguchi, Misa; Sebe, Mayu; Miyatake, Yumiko; Kitamura, Tadahiro; Tsutsumi, Rie; Harada, Nagakatsu; Nakaya, Yutaka; Sakaue, Hiroshi

    2016-01-01

    Leptin is a key regulator of energy intake and expenditure. This peptide hormone is expressed in mouse white adipose tissue, but hardly expressed in 3T3-L1 adipocytes. Using bisulfite sequencing, we found that CpG islands in the leptin promoter are highly methylated in 3T3-L1cells. 5-azacytidine, an inhibitor of DNA methyltransferase, markedly increased leptin expression as pre-adipocytes matured into adipocytes. Remarkably, leptin expression was stimulated by insulin in adipocytes derived from precursor cells exposed to 5-azacytidine, but suppressed by thiazolidinedione and dexamethasone. In contrast, adipocytes derived from untreated precursor cells were unresponsive to both 5-azacytidine and hormonal stimuli, although lipid accumulation was sufficient to boost leptin expression in the absence of demethylation. Taken together, the results suggest that leptin expression in 3T3-L1 cells requires DNA demethylation prior to adipogenesis, transcriptional activation during adipogenesis, and lipid accumulation after adipogenesis. PMID:27494408

  16. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  17. Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP).