Figure 2

PCAF is a co-activator to AR and promotes DHT-induced AR transcriptional activity and cell growth. A, knockdown of PCAF by siRNA and forced expression of PCAF through transfection of the pCX-PCAF in LNCaP cells, as confirmed by Western blot and PCR analysis. B, functional manipulation of PCAF on DHT-induced transcription of PSA in LNCaP cells. Cells were exposed to DHT (10 nM) for up to 48 h, followed by qRT-PCR. DHT was also used to stimulate LNCaP cells that were pre-treated with siRNA to PCAF or transfected with the pCX-PCAF for 24 h. C and D, functional manipulation of PCAF on DHT-induced PSA-6 kb luciferase activity in LNCaP cells. Cells were transfected with the PSA-6 kb luciferase reporter plasmid with PCAF siRNA or pCX-PCAF for 24 h, then exposed to DHT (10 nM) for 24 h. Luciferase activity was measured and presented as the ratio to β-gal. E and F, functional manipulation of PCAF on DHT-stimulated LNCaP cell growth. Cells were transfected with pCX-PCAF (E) or treated with PCAF siRNA (F) for 24 h and then exposed to DHT (10 nM) for 72 h, followed by MTS assay. G and H, ChIP analysis of DHT-induced promoter recruitment AR and PCAF to the ARE region of the PSA gene in LNCaP cells. Cells were exposed to DHT (10 nM) for 5 h and immunoprecipitated with antibodies to AR or PCAF, or the IgG control. Specific PCR primers covering the ARE-I region of the PSA promoter were used for the PCR analysis and data were presented as ratio to the input. Data in A to F are averages of three independent experiments. *, p < 0.05 compared to non-DHT treated cells (in B, C, D, E, F and H) or siRNA control (in A). ^#, p < 0.05 compared to cells treated with DHT only. Luc = luciferase activity.