Epigenetic alteration to activate Bmp2-Smad signaling in Raf-induced senescence

Figure 7 Knockdown of Bmp2 and overexpression of Smad6 inhibited growth arrest by Raf. A: Real-time-PCR for Bmp2 and Smad6, normalized to Ppib. Fold expression levels compared to RafV600E cells are shown. Knockdown of Bmp2 expression by shRNA and induction of Smad6 expression by retrovirus infection were confirmed. Mean and standard error in triplicated samples are shown; B: Growth curve of RafV600E + shBmp2 and RafV600E + Smad6 cells. While RafV600E cells showed growth arrest, Bmp2-knocked-down RafV600E cells (RafV600E + shBmp2, black line) and Smad6-overexpressed RafV600E cells (RafV600E + Smad6, black dotted line) showed continual growth and escape from Raf-induced senescence; C: Count of SA-β-gal(+) cells. Compared with RafV600E cells, Bmp2-knocked-down RafV600E cells (RafV600E + shBmp2) and Smad6-overexpressed RafV600E cells (RafV600E + Smad6) rarely showed SA-β-gal(+) cells. Mean and standard error in three repeated experiments are shown. SA-β-gal: Senescence-associated β-galactosidase.