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. 2012 Mar 5;7(3):e31052. doi: 10.1371/journal.pone.0031052

Figure 3. p21 is regulated by AR and required for cellular senescence.

Figure 3

AR-dependent senescence required increased p21. (A) PC3-AR cells were treated with Dox, DHT and Fl, at indicated combinations; cell extracts were analyzed by Western blot for p21, and ppRb. Tubulin was used as a loading control (A). Note the decrease in Rb phosphorylation and p21 increase in DHT-treated cells at every time point. (B) RWPE-1 cells, non-transfected or transfected with pLVX-AR or pLVX vector were treated with vehicle EtOH and DHT with or without Fl. Cell extracts were analyzed for AR, p21 and GAPDH to assess loading. (C) AR binding to p21 promoter: ChIP was performed with AR antibodies and AR-bound DNA amplified with the primers for the promoter region adjacent to the putative ARE element within p21 promoter (top) using real-time PCR. Each sample was run in triplicates, normalized per input DNA, and fold change in occupancy was calculated as FC = 2 (−ΔΔCT [exp-con]). Note increased AR binding to the p21 promoter in the presence of DHT and the reversal by flutamide (Fl, concentration shown in µM). The results of three independent experiments are pulled together (P<0.04). (D) PC3-AR cells were transfected with p21 or non-target control siRNA. Cell extracts were collected after 48 hours and analyzed by Western blot for p21, phospho-Rb (ppRb) and tubulin (loading control). Untransfected cells (-) are shown for comparison. (E, F) The transfectants were allowed 48 hours to recover and treated 5 days with Dox, DHT and Fl, as indicated; senescence was measured by SA-βGal assay. Note a significantly lower senescence levels after p21 knock-down (F, P<0.006). SA-βGal positive cells were quantified as above. Means and S.D. were calculated for three independent experiments. Representative images are shown (F).