The effect of 17-HASs on the levels of AR protein and AR mRNA.
A, AR protein from LNCaP cells cultured in charcoal-stripped serum (CSS) and treated with the indicated concentrations of TOK-001 (5–15 μm). Control (C) cells were treated with an equal volume of DMSO. A representative Western blot of whole cell extracts collected on day 1 or day 3 post-treatment. The levels of α-tubulin were also determined as a loading control. B, Western blot analysis for AR protein from LNCaP cells cultured in CSS and treated for 3 days with the indicated concentrations of abiraterone alcohol (5–15 μm). Control (C) cells were treated with an equal volume of ethanol. The levels of α-tubulin were also determined as a loading control. C, Western blot analysis for intracellular PSA protein levels in LNCaP cells cultured in FBS and treated for 3 days with the indicated concentrations of TOK-001 or abiraterone alcohol. Control (C) cells were treated with an equal volume of either DMSO or ethanol. The levels of α-tubulin were also determined as a loading control. D, recovery of AR protein expression after TOK-001 treatment. Western blot analysis of AR steady-state protein levels in LNCaP cells cultured in CSS and treated with DMSO (D) or 5 μm TOK-001 (+T) for 5 days (Days 0–5, left panel). Treated cells were then subcultured into fresh media without drug (-T) and incubated for an additional 5 days (Days 6–10, right panel). The levels of α-tubulin were also determined as a loading control. E, Western blot of AR protein levels in LAPC-4 cells cultured in CSS for 3 days with the indicated concentrations (0.25–2.5 μm) of TOK-001 and abiraterone alcohol. Control (C) cells were treated with an equal volume of either DMSO or ethanol. The levels of α-tubulin were also determined as a loading control. F, qRT-PCR of AR steady-state mRNA levels in LNCaP cells cultured in CSS and treated for 24 h with the indicated concentrations of TOK-001. AR mRNA levels were normalized to RPLPO. Data represent the mean and S.D. of independent experiments.