Figure 2

NPAS2 promotes HCC cell survival in vitro. (a) HCC cells were transiently transfected with siRNA or expression vector as indicated. Cells were reseeded for MTS cell viability assay 24 h after transfection. siNPAS2-1 and siNPAS2-2, siRNAs against NPAS2; siCtrl, control siRNA; NPAS2, expression vector encoding NPAS2; EV, empty vector. (b) Colony formation assay in HLE and HLF cells with treatment as indicated. (c) Cell proliferation ability was evaluated using EdU incorporation assay in HLE and HLF cells with treatment as indicated. Scale bar, 50 μm. (d) Flow cytometry analysis of apoptosis by Annexin V (an indicator of apoptosis) and PI staining in both HLE and HLF cells 48 h after transfection with siRNA or expression vector as indicated. HLF cells were also treated with CCCP (150 μ M) for 4 h before apoptosis analysis. (e) Western blot analyses for protein levels of Cyto C in cytoplasm and mitochondria of HLE and HLF cells with treatment as indicated. β-Actin and COX IV were used as loading controls for cytoplasm and mitochondria, respectively. Cyto, cytoplasm; Mito, mitochondria. (f) Western blot analyses for protein levels of NPAS2, cleaved caspase 9, cleaved caspase 3 and cleaved PARP in HLE and HLF cells with treatment as indicated. Experiments were repeated twice with independent cell extracts and representative data were presented. The data shown are the mean±S.E.M. from three independent experiments. *P<0.05; **P<0.01