Fig. 1: ARD1 knockdown in LNCaP inhibits endogenous AR nuclear translocation.

Nuclear translocation of AR proteins in LNCaP cells was analyzed by confocal fluorescence microscopy. The AR antibody (Santa Cruz Cat# SC-816) and the Alexa Fluor 488 secondary anti-rabbit IgG antibody (Cell Signaling Cat# 4412) were used to probe AR in PCa cells. LNCaP cells were cultured in RPMI-1640 medium with 10% FBS before knockdown. Lentivirus for ARD1 knockdown was generated using the Lipofectamine 3000 (ThermoFisher Scientific) system. The empty vector and knockdown cells were cultured in phenol red-free RPMI-1640 media supplemented with 10% charcoal stripped-FBS and were fixed with 4% paraformaldehyde. Nuclei were stained with 2.5 μM DRAQ5 (Cell Signaling Cat# 4084). N = 3, representative images shown. Confocal images were obtained using an Olympus Confocal Laser Microscope with a ×60 oil-immersion objective on a Z-stage