FIGURE 7.

X15695-mediated regulation of AR action in prostate cancer cells. A, Western blot analysis of BAG1 in LNCaP cells transduced with BAG-1 shRNA and control shRNA. Antibodies to vinculin, AR, tubulin, and GAPDH were used as controls. B, Quantification of the effect of X15695 on clonal expansion of control and BAG1 shRNA knockdown LNCaP cells. Each point represents the mean ± SEM. n = 3. C, Western blot analysis of AR and BAG1 in extracts of LNCaP and 22Rv.1 cells treated with the indicated concentrations of X15695 for 24 hours. β-actin was used as a loading control. D–F, GSEA plots of the topmost signaling pathways and heat maps of log₂ fold-change in gene expression in the comparison of DHT + X15695 versus DHT and X15695 versus vehicle in LNCaP cells. G, Quantitative RT-PCR was carried to detect the effect of X15695 on DHT- mediated expression of the indicated target genes in LNCaP cells. Cells were treated with vehicle or 10 nmol/L DHT and X15695 (5 μmol/L) for 16 hours. The results are the means ± SEM (n = 4; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant). H, 22Rv.1, LAPC-4, and LNCaP cells were transfected with control and p53 siRNA for 16 hours and treated with vehicle or X15695 (5 μmol/L) for 24 hours. Western blot analysis was performed with anti-p53 and anti-β-actin antibodies. I, MTT cell viability assay carried out with 22Rv.1, LAPC-4, and LNCaP cells previously transfected with control and p53 siRNA and treated with vehicle and X15695 for 72 hours. The results are the mean ± SEM (n = 3; *, P ≤ 0.05).