Fig. 4.

Regulation of senescence by TGF-β signaling is dependent on miR-29-induced inhibition of H4K20me3. a Western blot for the indicated proteins in MEFs grown in 3% oxygen or 20% oxygen. β-actin served as a loading control. b, c RT-qPCR analysis of miR-29 expression in the cells from a. d Western blot to assess total H4K20 methylation in serially passaged MEFs grown in 3% oxygen or 20% oxygen. β-actin served as a loading control. e MEFs grown in 3% oxygen and transferred to 20% oxygen (left) or MEFs grown in 20% oxygen and transferred to 3% oxygen (right) were treated with or without inhibitors. Protein changes were tested by western blot. H4 and β-actin served as loading controls. f Western blots showing altered H4K20 methylation in MEFs incubated with or without inhibitors upon knockdown of shCtrl, Suv4-20h1, Suv4-20h2 or both. g SA-β-gal staining showing alterations in the SA-β-gal signal in the cells from f. Scale bar, 20 μm. One-way ANOVA with Dunnett’s multiple comparison test analysis was performed. h Immunofluorescent staining showing the Mki67 signal in MEFs exposed to the same treatment as those shown in f. Scale bar, 5 μm. i Western blot for analyzing the indicated proteins in MEFs subjected to inhibition of miR-29 expression followed by TGF-β treatment. H4 and β-actin served as loading controls. The error bars represent the s.d. obtained from triplicate independent experiments. Two-tailed unpaired Student’s t-tests were performed, **p < 0.01, ***p < 0.001