Mibolerone

LNCaP cells were purchased from American Type Culture Collection (Manassas, VA), and C4-2 cells were purchased from Uro Corporation (Oklahoma City, OK). LAPC-4 cells were kindly provided by C. L. Sawyers. LNCaP and C4-2 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 U/ml penicillin, and 0.25 μg/ml amphotericin B. LAPC4 cells were maintained in Iscove’s Modified Dulbecco’s Media with 10% FBS. Cells were incubated at 37°C with 5% CO2. For androgen dose and time-course studies, cells were plated in medium with 10% charcoal-stripped serum (CSS). After 48 h, Mibolerone (mib), a potent synthetic androgen or ethanol (EtOH) was added. For protein and RNA synthesis inhibition, LNCaP cells were treated with Cycloheximide (CHX, 25 μg/ml) and actinomycin D (5 μM), respectively. Cycloheximide and actinomycin D were purchased from Sigma-Aldrich (St. Louis, MO). Mibolerone was purchased from Steraloids Inc (Newport, RI). MDV3100 (enzalutamide) was acquired from Medivation (San Francisco, CA). To measure the proliferation of C4-2 cells following androgen treatment, cells were treated with vehicle (ethanol) or 10 nM of Mibolerone for 24 h and cell number were measured by trypan blue assays (Mediatech, Inc, Manassas, VA).