Figure 5.

Effect of Hsp70 inhibition on AR transcriptional activity. A. C4–2 cells cultured in RPMI1640 with 5% CSS were treated with Hsp70 inhibitor VER-155008 at the indicated concentrations, in the presence of 1 nM R1881. Cell lysates were prepared 24 hours after the treatment and then analyzed by Western blotting using anti-AR, anti-PSA, and anti-GAPDH antibodies. B. Protein expression in C4–2 cells cultured in 5 % CSS RPMI 1640 media were treated with vehicle, 1 nM R1881, or 1 nM R1881 plus 15 μM VER-155008 for 72 hours, then analyzed by western blotting using anti-AR, anti-PSA, and anti-γ-Tubulin antibodies. C. Relative mRNA expression in C4–2 cells treated as in B, then harvested for quantitative real-time PCR analysis. D. C4–2 cells stably transfected with both the PSA-luciferase plasmid and Renilla plasmid (47) were treated with either VER-155008 or another Hsp70 inhibitor, 18BBQU (UPCMLD18BBQU015254), in the presence of 1 nM R1881 for 24 hours. Luciferase assays were performed as described previously (66). To verify 18BBQU impact on AR protein level, C4–2 cells treated with 18BBQU in parallel were tested by immunoblotting using anti-AR antibody (Figure S4).