Figure 5. p63 expression is blocked by P21 and contributes to AR-dependent senescence.

This figure illustrates the link between p21 and p63 n AR-driven senescence. (A) PC3-AR cells were transfected with non-target (NT), p21 or p63 siRNA. Dox ± DHT were added to the media 24 hours post-transfection. Whole cell lysates were collected after 3 days and p63 expression measured by Western blot. Arrows point to TAp63-α and ΔNp63; * indicates non-specific band. Note the lack of p63 downregulation in p21 knock-down cells. (B) RWPE-1 cells were transfected with pLVX-AR or control pLVX and treated with vehicle EtOH or DHT. TAp63-α and ΔNp63 were measured as in (A). (C) PC3-AR cells were transfected with p21 or non-target (NT) siRNA. The cells were placed in Dox, ± DHT. RNA was isolated 3 days post-transfection. P63 message was measured by real-time RT-PCR. Note an approximately seven-fold reduction upon DHT treatment and increased expression after p21 knock-down. (D) In situ immunofluorescence of PC3-AR cells with pan-p63 antibody. Note pronounced p63 nuclear staining in Dox-treated cells, the lack of nuclear staining after 3 days in DHT and restored nuclear staining in the presence of Fl (top). Below: P63 nuclear localization (green pseudocolor), nuclei are counterstained with DAPI (red pseudocolor); colocalization appears in yellow. (E) PC3-AR cells were transfected with p21, p63 or control siRNA, cultured 48 hours in Dox ± DHT and stained with p63 antibodies. Note similar, diffuse p63 localization with the nuclear presence (filled arrows) in Dox-treated cells, the lack of nuclear localization in the presence of DHT (empty arrows), which is lost after p21 knock-down (filled arrows). P63 knock-down results in a weak, residual cytoplasmic staining. (F, G) Parental PC3 cells lacking AR expression were transfected with NT and p63 siRNA, cultured 5 days and senescence measured by SA-βGal assay. Representative images (left) and quantitative analysis (right) are shown. * indicates P<0.05.