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  • Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts. 23919723

    Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4(-/-)), we transfected Klf4(-/-)MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4(+/+)) and untransfected or mock-transfected Klf4(-/-)MEFs.We show that overexpression of Klf4 in Klf4(-/-)MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Mammalian target of rapamycin complex I (mTORC1) activity in ras homologue enriched in brain (Rheb)-deficient mouse embryonic fibroblasts. 24303063

    The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.
    Document Type:
    Reference
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    Multiple
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    Multiple

  • Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts. 25230394

    We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Effect of BIX-01294 on H3K9me2 levels and the imprinted gene Snrpn in mouse embryonic fibroblast cells. 26285804

    Histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation is thought to be a major influential factor in cellular reprogramming, such as somatic cell nuclear transfer (SCNT) and induction of pluripotent stem cells (iPSCs). The diazepin-quinazolin-amine derivative (BIX-01294) specifically inhibits the activity of histone methyltransferase EHMT2 (euchromatic histone-lysine N-methyltransferase 2) and reduces H3K9me2 levels in cells. The imprinted gene small nuclear ribonucleoprotein N (Snrpn) is of particular interest because of its important biological functions. The objective of the present study was to investigate the effect of BIX-01294 on H3K9me2 levels and changes in Snrpn DNA methylation and histone H3K9me2 in mouse embryonic fibroblasts (MEFs). Results showed that 1.3 μM BIX-01294 markedly reduced global levels of H3K9me2 with almost no cellular toxicity. There was a significant decrease in H3K9me2 in promoter regions of the Snrpn gene after BIX-01294 treatment. A significant increase in methylation of the Snrpn differentially methylated region 1 (DMR1) and slightly decreased transcript levels of Snrpn were found in BIX-01294-treated MEFs. These results suggest that BIX-01294 may reduce global levels of H3K9me2 and affect epigenetic modifications of Snrpn in MEFs.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Novel role of base excision repair in mediating cisplatin cytotoxicity. 21357694

    Using isogenic mouse embryonic fibroblasts and human cancer cell lines, we show that cells defective in base excision repair (BER) display a cisplatin-specific resistant phenotype. This was accompanied by enhanced repair of cisplatin interstrand cross-links (ICLs) and ICL-induced DNA double strand breaks, but not intrastrand adducts. Cisplatin induces abasic sites with a reduced accumulation in uracil DNA glycosylase (UNG) null cells. We show that cytosines that flank the cisplatin ICLs undergo preferential oxidative deamination in vitro, and AP endonuclease 1 (APE1) can cleave the resulting ICL DNA substrate following removal of the flanking uracil. We also show that DNA polymerase β has low fidelity at the cisplatin ICL site after APE1 incision. Down-regulating ERCC1-XPF in BER-deficient cells restored cisplatin sensitivity. Based on our results, we propose a novel model in which BER plays a positive role in maintaining cisplatin cytotoxicity by competing with the productive cisplatin ICL DNA repair pathways.
    Document Type:
    Reference
    Product Catalog Number:
    MABE416
    Product Catalog Name:
    Anti-Cisplatin DNA Adducts Antibody, clone ICR4

  • Establishment of clinically compliant human embryonic stem cells in an autologous feeder-Free system. 21561302

    Applications of human embryonic stem cells (hESCs) are limited by the use of mouse embryonic fibroblasts feeder and animal-derived components during culture. In this study, we demonstrated the potential use of extracellular matrix (ECM) derived from the autologous feeders to support long-term undifferentiated growth of hESCs in xeno-free, serum-free, and feeder-free conditions. Autologous H9 ebF (feeder cells derived from outgrowth of embryoid body [EB] predifferentiated from H9 hESCs) was derived from EBs predifferentiated from H9 hESCs through a direct-plating outgrowth system. The ECM comprising collagen VI, laminin, and fibronectin was extracted from H9 ebF through a freeze-thaw procedure. The autologous ECM together with animal component-free TeSR™2 medium was used to support long-term growth of H1 and H9 hESC lines for up to 20 passages. The maintenance of hESC undifferentiated state by autologous ECM was confirmed by the positive staining of hESC-specific markers (Oct4, SSEA-4, and Tra-1-60) and the expression of pluripotency marker genes (Oct4, Nanog, and Sox2). Flow cytometry further showed that more than 99% of hESCs retained the expression of SSEA-3/4 after long-term culture on autologous ECM. Pluripotency of hESCs on ECM was further proven by in vitro EB formation and in vivo teratoma assay. Overall, this study suggested a strategy for efficient propagation of clinically compliant hESCs in an autologous feeder-free culture system.
    Document Type:
    Reference
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    Multiple
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    Multiple

  • Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts. 18571647

    The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1-8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.
    Document Type:
    Reference
    Product Catalog Number:
    07-352
    Product Catalog Name:
    Anti-acetyl-Histone H3 (Lys9) Antibody

  • ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment. 24595357

    We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1-/- and ROCK2-/- MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1-/- MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
    Document Type:
    Reference
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    Multiple
    Product Catalog Name:
    Multiple

  • Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition. 19775255

    Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV-infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV-like particles containing a beta-lactamase-VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte-derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre-treated for 48 h with TNFalpha were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNFalpha modulates cathepsin B activity and affects EBOV entry into and infection of human DCs.
    Document Type:
    Reference
    Product Catalog Number:
    AB3738

  • B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts. 15199148

    Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.
    Document Type:
    Reference
    Product Catalog Number:
    07-583