Enzalutamide

In 8-well chamber slides (BD Bioscience, Shanghai, China, catalog number: 354108), 30 µL growth factor reduced BD Matrigel (BD Biosciences, catalog number: 354230) was added per well. Slides were placed in a cell culture incubator for at least 15 min to solidify the Matrigel. Next, 2000 C4-2B cells/well or 4000 LNCaP cells/well were seeded in RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide (Beyotime Biotechnology) and 2% Matrigel. Media were replenished every 3 days. Chamber slides were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, and then photographed. Image J program (NIH, USA) was used to measure the diameter of each sphere. Spheres with a diameter larger than 80 μm were counted.

LNCaP and C4-2B cells were transiently transfected with pGL3-Basic, pGL3-KLF5, or pGL3-PSA, or pGL3-MMTV and pRL-TK (Renilla luciferase, Promega, Madison, WI) as an internal control. After 48 h of transfection and enzalutamide (Beyotime Biotechnology, Shanghai, China, catalog number: SC0074) treatment, cells were lysed in 5 × lysis buffer (Promega) for 30 min and luciferase activity was measured using a luminometer (Tristar LB941, Berthold Technologies, BadWild, Germany). Firefly luciferase activity was normalized to Renilla luciferase activities in each reaction. Experiments were performed in triplicate.

Human PCa cell line LNCaP was purchased from ATCC, and C4‐2B was a gift from Dr. Leland Chung of Cedar Sinai Medical Center. Both cell lines were cultured in RPMI‐1640 medium supplemented with 10% foetal bovine serum (FBS; Gibco) in a humidified incubator (37°C and 5% CO₂). For experiments involving the treatment of R1881 (Melonepharma, Dalian, China), cells were incubated in phenol red‐free medium containing 5% charcoal‐stripped FBS for 24 or 72 h, except the experiment involving a combined treatment of R1881 and enzalutamide (Beyotime), in which complete medium was used.
For gene silencing by RNA interference (RNAi), cells were transiently transfected with siRNAs using the Lipofectamine RNAiMAX reagent (Invitrogen). The siRNA sequence against human AR (siAR) was 5′‐CAAGGGAGGUUACACCAAA‐3′, validated in a previous study.²¹