Abiraterone

The SLCO1B3 3′UTR was synthesized with flanking NotI and XhoI restriction sites in a pUC57 vector by GENEWIZ, Inc (South Plainfield, NJ). The synthesized sequence was then cloned into complementary restriction sites in a psiCHECK-2 vector, which contains both renilla and firefly luciferase reporter genes (Promega, CAT#: C8021). The constructed plasmid was sequenced to verify successful cloning.
22Rv1 cells were seeded in a 96-well dish for 24 h before transfection with 100 ng of psiCHECK-2 empty vector or 3′UTR plasmid using Lipofectamine 2000 (Life Technologies, Cat#: 11668019). The following day, cells were treated with 20 µM abiraterone (SelleckChem, Cat#: S1123) or vehicle control (0.4% DMSO) (Sigma Aldrich). After a 24-h treatment period, samples were lysed and assayed using the Dual-Luciferase Reporter Assay System (Promega, Cat#: E1910) per manufacturer’s protocol. Raw luminescence was measured for Renilla and firefly reporters using SpectraMax iD3 (Molecular Devices). For microRNA mimic treated samples, cells were instead co-transfected for 24 h with the psiCHECK-2 plasmid or empty vector and 1 pmol of hsa-miR-579-3p miRvana mimic or negative control (Life Technologies, Cat#: 4464066) before performing the reporter assay.

For in vitro studies, enzalutamide and abiraterone (Selleck Chemicals, Houston, TX) were dissolved in DMSO (vehicle, Sigma Aldrich, St. Louis, MO) to a concentration of 10 mM and stored at −20°C. A concentration of 10 μM of either enzalutamide or abiraterone was used for all in vitro experiments where media and drug or vehicle were replaced daily.

A172 and T98G cells were purchased from ATCC (Manassas, VA, USA), and all cell lines used in this study were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, as reported previously [7 (link),8 (link),32 (link)]. Abiraterone was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO, MilliporeSigma Corporate, St. Louis, MO, USA). Hydrogen peroxide solution, antimycin a (AMA), and tert-butyl hydroperoxide (TBHP) were purchased from MilliporeSigma Corporate (St. Louis, MO, USA). DDK (Flag)–Myc–CYP17A1 was purchased from OriGene Technologies, Inc. (Rockville, MD, USA). Flag-SAR1a and -SAR1b constructs were purchased from GenScript^® (Piscataway, NJ, USA).

Abiraterone and enzalutamide were purchased from Selleckchem (Houston, TX). Galeterone, finasteride, and ketoconazole were purchased from Sigma-Aldrich (St. Louis, MO). VT-646 (seviteronel) was ordered from Chemscene (Monmouth Junction, NJ). Abiraterone acetate for xenograft studies was purchased from Medkoo Biosciences (Morrisville, NC). All drugs were dissolved in DMSO, aliquoted and stored at − 80 °C. Testosterone (Sigma) was dissolved in ethanol and DHT (Steraloids Inc., Newport, RI) was dissolved in DMSO.