(A) Suppression of AR-collaborating factor levels by RNAi. LNCaP cells were transfected with siRNA targeting each factor and a control siRNA. Forty-eight hours posttransfection, cells were treated with or without 100 nM DHT for 16 hr, and western blots were performed using the antibodies indicated.
(B) Effects of siRNA on PSA, TMPRSS2, and GADPH gene expression. Forty-eight hours after siRNA transfection, cells were treated with or without 1 or 100 nM DHT for 4 and 16 hr. Total RNA was isolated and amplified by real-time RT-PCR using transcript-specific primers (Table S1). The no-ligand control was measured at 4 hr.
(C) Effects of silencing GATA2 and Oct1 on AR, Pol II, Oct1, and GATA2 recruitment to the PSA and TMPRSS2 enhancers. AR, Pol II, Oct1, and GATA2 ChIPs were performed after vehicle or 4 hr 100 nM DHT treatment of siLuc, siGATA2, or siOct1-transfected cells. Graphical representations of the mean ± SE of two to three independent experiments are shown in (B) and (C).