Fig. 2.

miR-29-mediated reduction of H4K20me3 leads to premature cellular senescence. a, b Luciferase assays with the wild-type Suv4-20h1 3′ UTR or mutated Suv4-20h1 3′ UTR (a), as well as with the wild-type Suv4-20h2 3′ UTR or mutated Suv4-20h2 3′ UTR (b) in the predicted binding site of miR-29a, miR-29b and miR-29c, transfected with control miRNA mimics (M-miR-Ctrl) or miR-29 mimics (M-miR-29a, M-miR-29b and M-miR-29c). c Protein levels of H4K20 were measured by western blotting in MEFs transfected with M-miR-29 or miR-29 inhibitors (I-miR-29a and I-miR-29c). β-actin served as a loading control. d Western blot for global H4K20 methylation in cells with lentivirus-mediated ectopic expression of the indicated miR-29. β-actin served as a loading control. e SA-β-gal staining and diagrams showing MEFs from PD4 transfected with M-miR-29 and MEFs from PD8 transfected with I-miR-29. Scale bar, 20 μm. One-way ANOVA with Dunnett’s multiple comparison test was performed. f, g Immunofluorescent staining of Mki67 in MEFs transfected with M-miR-29 (f) or I-miR-29 (g). Scale bar, 5 μm. h Cells were first transfected with M-miR-Ctrl, M-miR-29a or M-miR-29c, after which they were infected with pBabe, pBabe-Suv4-20h1 or pBabe-Suv4-20h2 and subjected to western blotting for the indicated protein. β-actin served as a loading control. i SA-β-gal staining analysis was performed as described for cells in h. Scale bar, 20 μm. j Immunofluorescent staining of Mki67 in the cells from i. Scale bar, 5 μm. The error bars show the s.d obtained from triplicate independent experiments. Two-tailed unpaired Student’s t-tests were performed, **p < 0.01, ***p < 0.001