Figure 6

Downregulation of ASCL1 is important for TGF-β-mediated apoptosis in SCLC cells. (a) qRT-PCR analysis shows ASCL1 expression. H345 cells were infected with lentivirus vectors encoding control shRNA (shNTC) or shRNA that targeted ASCL1 (shASCL1). H345-TβRII cells stimulated with TGF-β for 48 h served as control. Data represent mean±s.d. *P<0.05; ***P<0.001. (b) Cell proliferation assay. Cells in (a) were incubated for 12 days. Data represent mean±s.d. ***P<0.001. (c) Cell cycle analysis of cells in (b). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (d) Immunoblot of cell lysates probed with the indicated antibodies. H345-GFP and H345-TβRII cells were infected with lentivirus vectors encoding GFP alone or ASCL1, and then stimulated with TGF-β for 12 days. (e) Cell cycle analysis of cells in (d). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of each cell cycle stage in indicated cells is shown. (f) qRT-PCR analysis shows ASCL1 expression in H345 cells transfected with control siRNA (siNTC) or siRNA that targeted ASCL1 (siASCL1). ASCL1 expression was determined 72 h post-transfection. Data represent mean±s.d. ***P<0.001. (g) Mice received subcutaneous transplants of cells in (f) (siNTC, n=7, siASCL1, n=7). (Left panels) Representative photographs; (right panel) incidence of tumor formation 2 weeks after injection. Arrow heads indicate tumors. GFP, green fluorescent protein; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR.