FIG 1.
Knockdown of p16INK4A induces senescence of HeLa cells. (A) HeLa cells were transfected with p16INK4A siRNA or control siRNA (200 pmol per well) for three times in total (day 1, day 4, and day 7). p16INK4A knockdown was verified by Western blotting (inset). Cells were counted, and a growth curve was established. Shown are the results (mean ± standard deviation [SD]) from three independent experiments. cPDL, cumulated population doublings. (B) At day 12, cells were assessed for presence of SA-β-gal activity. Bars represent the relative percentage of SA-β-gal-positive cells (mean ± SD) observed within three visual fields from three independent experiments. Two representative micrographs are also shown. (C) HeLa cells were treated as described for panel A and harvested at day 10 posttransfection. Left panel, extraction of total RNA was followed by subsequent cDNA synthesis, and mRNA levels of B2M and p21WAF-1/Cip-1 were assessed by qRT-PCR. p21WAF-1/Cip-1 mRNA levels were normalized to B2M mRNA. Shown are the results (mean ± SD) of three independent experiments. Right panel, cell extracts were subjected to SDS-PAGE and analyzed by Western blotting using antibodies for the detection of p21WAF-1/Cip-1, p53, and tubulin, as indicated. Extracts of cisplatin-treated human fibroblasts were added as a positive control for the expression of p21WAF-1/Cip-1 and p53; as a further control, extracts of untreated HeLa cells were used. Three independent experiments were performed, and results of one representative experiment are shown. (D) Left panel, HeLa cells were treated as for panel A, stained with annexin V, and analyzed by flow cytometry, and the relative percentage of annexin V-positive cells was determined. Shown are the results (mean ± SD) of three independent experiments. Right panel, HeLa cells were treated as for panel A, and caspase-3/7 activity was determined and displayed as relative luminescence units (RLU). Staurosporine-treated cells were used as a positive control for the assay. Shown are the results (mean ± SD) of three independent experiments. n.s., not significant.