Figure 4

EZH2-mediated silencing of TβRII is required for SCLC tumor formation. (a) qRT-PCR analysis post-immunoprecipitation with anti-EZH2 antibody shows EZH2 enrichment in the TGFBR2 locus of H345 cells. Hypoxanthine guanine phosphoribosyl transferase1 (HPRT1) was used as the negative control. Data represent mean±s.d. TGFBR2 locus 1, chromosome 3: 30606416–30606492 bp; TGFBR2 locus 2, chromosome 3: 30605676–30605771 bp; TGFBR2 locus 3, chromosome 3: 30604616–30604714 bp; TGFBR2 locus 4, chromosome 3: 30603967–30604061 bp. *P<0.05; **P<0.01. (b) qRT-PCR analysis shows TGFBR2 expression. SCLC cells were treated with GSK343 (10 μM) for 7 days. Data represent mean±s.d. (c) qRT-PCR analysis shows EZH2 and TGFBR2 expression. H345 cells were infected with lentivirus vectors with control shRNA (H345-shNTC) or shRNA that targeted EZH2 (H345-shEZH2). Data represent mean±s.d. ***P<0.001. (d) Immunoblot of cell lysates from cells in (c) stimulated with TGF-β for 2 h and probed with the indicated antibodies. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. (e) qRT-PCR analysis shows SMAD7 expression. Cells in (c) were stimulated with TGF-β for 4 h. Data represent mean±s.d. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. **P<0.01; ***P<0.001. (f) Cell cycle analysis of cells in (c) stimulated with TGF-β for 12 days. (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. (g) Mice received subcutaneous transplants of H345-shNTC cells (n=7) or H345-shEZH2 #1 cells (n=7). (left panels) Representative photographs 4 weeks after injection. Arrow heads indicate tumors. (right panel) Tumor volumes at the indicated time points. Data represent mean±s.e.m. **P<0.01. SCLC, small cell lung cancer; qRT-PCR, quantitative real-time reverse transcription-PCR.