Figure 1. AR activation causes cell cycle arrest and senescence in PC3 cells.

AR activation causes cell cycle arrest and senescence in PC3 cells. (A) In situ immunofluorescence with anti-AR antibody: note diffuse AR staining in control treated PC3-AR cells (left) and nuclear translocation on 3 day of DHT treatment (right). (B) Time-dependent inhibition of cell growth in vitro. PC-3 cells were cultured in Dox ± DHT; viable cells were measured using WST-1 reagent. Each time point represents mean ± standard deviation of three independent experiments. The difference between DHT treatment (red) and control EtOH (green) is statistically significant on day 5 (P<0.01). (C) Cell cycle analysis: the cells were grown in Dox, in the absence (D) and in the presence of DHT (DD) for 3 and 5 days; after 7 days the cells were passed (P1) and incubated for another 5 days. Cell cycle distribution was analyzed by flow cytometry. Note increased cell number in Go/G1 phase accompanied by decreased in S and G2/M populations in DHT-treated cells (DD) compared to Dox alone (D). (D) PC3-AR cells were cultured in Dox and treated with DHT ± Flutamide (Fl, 20 µM). After five days senescence was measured using SA-βGal assay. Note increased βGal positivity upon AR activation and the lack of senescence in the presence of Fl. (E) Senescent cells were counted on the digital images of 5 random fields using Image Tool 3.00 software (UTHSCSA); means of three independent experiments with S.D.M. are shown. (F) RWPE-1 cells were transfected with control lentivirus or lentiviral vector encoding AR. AR expression was measured in whole cell lysates by Western blot. GAPDH antibody was used to assess loading. (G, H) RWPE-AR and vector transfected (RWPE-C) cells were cultured in DHT, DHT and Flutamide (Fl) or with equal volume of EtOH added as control. Senescence was assessed with SA-βGal assay (I) and quantified as in (E).