AR Signaling Pathway Regulation

AR negative cell lines (M12, PC3 and LNCaP^APIPC) (see the Supplementary Data for details) were co-transfected with the p3XFLAG-CMV10-based expression vector and the reporter plasmid. Specifically, the reporter assay was done with either the luciferase reporter driven by canonical androgen responsive regions (pARR2PB-luc),²³ or a luciferase construct under the control of AR-V-specific binding sites derived from the promoter element of the ubiquitin-conjugating enzyme E2C (UBE2C) gene (UBE2C-luc).^{24 (link), 25 (link), 58 (link)}Renilla luciferase expression plasmid was used as an internal standard in conjunction with pARR2PB-luc. At 16 h after transfection, cells were treated with 1 nm 5α-dihydrotestosterone (DHT) or vehicle control. As necessary, 10 μm enzalutamide was added 2 h prior to DHT treatment. Luciferase assay was done at 24 h after DHT treatment using the Dual-Glo luciferase assay system (Promega, Madison, WI, USA) with GloMax Multi Detection System for detection (Promega). Firefly luciferase luminescence was normalized to that of the co-expressed Renilla luciferase or protein concentration per each sample.