Androgen stimulation

For short-term comparison of androgen response, LNCaP cells were subcultured to 50% confluence in phenol red-free RPMI-1640 supplemented with 1% charcoal stripped FBS (CS-FBS) for 48 h (androgen-depleted conditions). DHT was serially diluted into media from concentrated stocks solubilized in ethanol as a vehicle. Androgen-depleted media were replaced with media containing the indicated concentration of DHT for 48 h, and harvested for analysis. For long-term selection, cells were grown for 10 days in phenol red-free RPMI-1640 media containing 5% CS-FBS and 0 (vehicle), 0.1, 1, or 10 nM DHT. Media were renewed every two days. Following selection, cells were either maintained in these conditions, or subjected to 48 h in androgen-depleted media followed by 24–48 h stimulation with 10 nM DHT to quantify the magnitude of the androgen-stimulatable response.