Figure 4: Androgen stimulation increases AR occupancy at target genes and upregulates AR-target gene expression in AA PCa cells. (a) AA PCa cell line MDA PCa 2b and (b) AA PCa cell line E006AA were treated with 100 nM DHT for 18 hr (top panels). Representative ChIP-PCR assays confirmed DHT-induced increases in AR occupancies at the promoter regions of RHOA, ITGB5, and PIK3CB genes. AR occupancies and relative gene expressions with or without androgen stimulation (bottom panels) were measured as a percentage of ChIP-to-Input signal and mRNA levels, respectively. KLK3 (PSA gene) and ACTB were used as positive and negative controls for the ChIP-PCR assays, respectively. For qRT-PCR, the relative expression levels of RHOA, ITGB5, PIK3CB, and KLK3 were determined by the ΔΔCt method using EIF1AX and PPA1 as endogenous genes for normalization. Cells were treated with a vehicle (<0.01% ethanol final concentration) or DHT for 18 hr prior to ChIP and qRT-PCR assays. Data are represented as the mean ± SEM of 3–5 independent ChIP and PCR experiments. *Significantly different AR occupancies and expression levels of AR-target genes in both cell lines ( using Student’s t-test).