Dihydrotestosterone

Reagents were of ACS grade or higher and were purchased from Fisher Scientific (Pittsburgh, PA, USA) and used without further purification. [1β-³H(N)]-Δ⁴-androstene-3,17-dione (4-AD) (Specific Radioactivity 96.2 Ci/mole) and [³H-R1881 (85.1 Ci/mmole) were obtained from Perkin-Elmer. 4-Androstene-3,17-dione and dihydrotestosterone were purchased from Steraloids and Sigma, respectively. Unlabeled R1881 was obtained from Perkin-Elmer. Nicotinamide adenine dinucleotide, Grade I reduced form (NADH) and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) were obtained from Roche Diagnostics (Indianapolis, IN, USA). R-biaclutamide was purchased from Sigma; Enzalutamide was obtained from Selleckchem; and abiraterone was purchased Euroasia Chemicals (Mumbai - 400 013, India ). Charcoal dextran stripped fetal bovine serum (CD-FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA).

SH-SY5Y cells were grown in DMEM-F12 (Sigma) medium supplemented with 10% heat-inactivated fetal bovine serum at 37 °C with 5% CO₂. Cells were plated in 24-well plates at a density of 1 × 10⁵ cells/well and cultured overnight before transfection. Transient transfection for luciferase activity assays was performed by lipofectamine 2000 (Invitrogen, CA, USA). Plasmids used were: pGL3-MOR (480 ng); pRL-CMV encoding renilla luciferase (20 ng); pFLAG-AR or empty pFLAG-CMV vector (200 ng). Twenty-four hours after transfection, the cells were treated with either 100 nM dihydrotestosterone (Steraloids, RI, USA) or testosterone (Sigma–Aldrich) for 24 h. Activity of the firefly and renilla luciferase in extracts of the transfected cells was determined using the Dual-Luciferase Reporter Assay System (Promega, WI, USA). For each sample, the activity of the firefly luciferase was divided by the activity of the renilla luciferase to correct for transfection efficiency. The corrected firefly luciferase activity of experimental constructs was expressed relative to that of the β-actin promoter-driven firefly luciferase. At least three independent experiments in triplicate were performed.