Figure 5. SIRT1 depletion increases the sensitivity of AR-dependent gene transcription and cell proliferation to DHT.

A) SIRT1 knockdown increases the sensitivity of AR-dependent gene transcription to DHT. Parental empty-vector transfected LNCaP cells (Ctrl) or LNCaP cells in which SIRT1 had been stably knocked down by siRNA expression (RNAi) were cultured in charcoal-stripped serum, transfected with the PSA-LUC reporter vector, and then treated with nicotinamide (NAM) or vehicle (Control), and exposed to the indicated concentrations of DHT (0–1 nM). Cells were harvested after 48 hr and assayed for luciferase activity. B) Immunoblot analysis of SIRT1, AR and β-actin levels in control-transfected LNCaP cells (Ctrl) and three LNCaP lines in which SIRT1 had been knocked down by stable expression of siRNA (RNAi-1, RNAi-2 and RNAi-3). C) SIRT1 knockdown increases the sensitivity of the proliferative response of LNCaP cells to DHT. Control, empty-vector transfected LNCaP cells (Ctrl) or LNCaP cell lines in which SIRT1 had been stably knocked-down by siRNA expression (lines RNAi-1, RNAi-2 and RNAi-3) were made quiescent by culture in charcoal-stripped serum, and exposed to the indicated concentrations of DHT (0–10 nM). Viable cells were quantitated at 72 hr by MTT assay, and the results expressed relative to values obtained from plates cultured without added DHT (assigned an arbitrary value of 1). In all relevant figures, relative luciferase activities were normalized to β-gal activity to control for transfection efficiency. The error bars represent the SEM. Asterisks (*) indicates significant differences between two groups (p<0.05).