SIRT1, Sp1, and CDK6 are potentially involved in miR-22–mediated cellular senescence. (A) Representative growth curves corresponding to MDA-D3 cells transfected with cont siR or siRNAs against SIRT1, Sp1, and CDK6. Each value was determined in triplicate. (B) The histograms display the percentage of SA-β-gal–positive cells in MRC-5 and MDA-D3 cells at day 6 after siRNA transfection. (C–F) Western blot analysis was performed in those cells as indicated in each panel at 72 h after siRNA/miRNA transfection (C–E) or day 6 after infection (F). β-actin was used as a loading control and the relative density of bands was densitometrically quantified. (G and H) SiHa cells were first transfected with miR-22 duplex and, 24 h later, sequentially transfected with expressing plasmids or empty vectors as indicated. Cell proliferation (G) and SA-β-gal (H) activity was evaluated at day 6 after miRNA transfection. Data in all the panels represent mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01.