Project description:We previously reported that human T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein constitutively activates TAK1. Here, we established Tax-positive HuT-102 cells stably downregulated TAK1 expression by short-hairpin RNA (HuT-shTAK1 cells), and investigated the physiological function of TAK1. Microarray analysis demonstrated that several interferon (IFN)-inducible genes including chemokines such as CXCL10 and CCL5 were significantly downregulated in HuT-shTAK1 cells. In contrast, Tax-mediated constitutive activation of NF-kB was intact in HuT-shTAK1 cells. IRF3, a critical transcription factor in innate immunity to viral infection, was constitutively activated in a Tax-dependent manner. Activation of IRF3 and IRF3-dependent gene expression were dependent on TAK1 and TBK1. On the other hand, IRF4, another IRF family of transcription factor overexpressed in a Tax-independent manner, negatively regulated the TAK1-dependent IRF3 transcriptional activity. Together, HTLV-1 manipulates IFN signaling by regulating both positive and negative IRFs. HuT-102 cells, a human cutaneous T cell lymphoma, were stably transfected with shRNA expression vectors against human MAP3K7 (TAK1) or firefly luciferase (Luc). The cells were maintained in media containing 0.5 mg/ml G418. For the experiment, the cells were incubated in media without G418 for 36 h at 37M-BM-0C. Total RNA samples were prepared from the cells. Gene expression was analyzed by an Affymetrix GeneChipM-BM-. system with a Human Genome U133-plus 2.0 array for analysis of over 47,000 transcripts. Sample preparation for array hybridization was carried out as described in the manufacturerM-bM-^@M-^Ys instructions. Two replicates per sample type.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:The microarray analysis showed an interesting up-regulation in the set of genes controlling the development of Th1, mainly IFN-gamma, and other type 1 interferon response genes including CXCL10 in IRF4 knocked-down HuT-102 cells. HuT-102 cells, a human cutaneous T cell lymphoma, were transiently transfected with siRNAs against human IRF4 or firefly luciferase. Cells were incubated for 60 h at 37 centigrade. Total RNA samples were prepared from the cells. Gene expression was analyzed by an Affymetrix GeneChip® system with a Human Genome U133-plus 2.0 array for analysis of over 47,000 transcripts. Sample preparation for array hybridization was carried out as described in the manufacturerâs instructions.
Project description:We previously reported that human T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein constitutively activates TAK1. Here, we established Tax-positive HuT-102 cells stably downregulated TAK1 expression by short-hairpin RNA (HuT-shTAK1 cells), and investigated the physiological function of TAK1. Microarray analysis demonstrated that several interferon (IFN)-inducible genes including chemokines such as CXCL10 and CCL5 were significantly downregulated in HuT-shTAK1 cells. In contrast, Tax-mediated constitutive activation of NF-kB was intact in HuT-shTAK1 cells. IRF3, a critical transcription factor in innate immunity to viral infection, was constitutively activated in a Tax-dependent manner. Activation of IRF3 and IRF3-dependent gene expression were dependent on TAK1 and TBK1. On the other hand, IRF4, another IRF family of transcription factor overexpressed in a Tax-independent manner, negatively regulated the TAK1-dependent IRF3 transcriptional activity. Together, HTLV-1 manipulates IFN signaling by regulating both positive and negative IRFs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.