Project description:We use lentivirus carrying shRNA for THADA to evaluate the efect of THADA knockdown in Huh7 cells. The pGIPZ shRNA plasmid specific to human THADA (clone ID V3LHS_318037 Open Biosystems, Inc.) or the pGIPZ non-silencing shRNAmir lentiviral plasmid (as a control) was cotransfected with the pPACKH1 plasmid mix in HEK 293 cells. Stable clones of Huh7 cells with THADA knockdown were routinely maintained in a medium containing of puromycin (500ng/ml)
Project description:To identify CD24 signaling pathway Affymetrix Human Genome U133 Plus GeneChip 2.0 shRNA CD24 (knockdown) or non-target control (NTC) was stably transduced into Huh7 HCC cells by lentiviral approach
Project description:E2F6, a potent transcriptional repressor, plays important roles in cell cycle regulation. However, roles of E2F6 in hypoxia-induced apoptosis are unknown. Here, we demonstrated biological functions of E2F6 in hypoxia-induced apoptosis and regulatory pathways. During hypoxia (CoCl(2), 800 microM)-induced human embryonic kidney 293 cell apoptosis, E2F6 expression was down-regulated with concurrent increases in E2F1 expression and transactivation. E2F6 overexpression abrogated hypoxia-induced apoptosis and alteration of E2F1. Conversely, specific knockdown of E2F6 by small interfering RNA had opposite effects. Chromatin immunoprecipitation assay confirmed that E2F6 regulated E2F1 expression through the transrepression of E2F1 promoter. Interestingly, E2F1 transactivation and apoptosis induced by hypoxia in cells stably expressing E2F1 were inhibited by E2F6 overexpression, suggesting that the inhibitory effects of E2F6 are not only mediated by the repression of E2F1 promoter. This was confirmed by E2F6-inhibited transactivation of E2F1 and apoptosis via competing with E2F1 for DNA binding sites evidenced by the different behaviors of E2F6DeltaC (C-terminal deletion) and E2F6.E68 (mutant DNA binding site) and by the lack of association of E2f6 with E2F1 protein. Moreover, hypoxia up-regulated expression of E2F1-responsive proapoptotic gene apoptosis protease-activating factor 1 was repressed by E2F6 overexpression. Together, these findings demonstrate a novel role of E2F6 in control of hypoxia-induced apoptosis through regulation of E2F1.