Fig 1: ARD1 nuclear localization in LNCaP and E006AA-hT cells.ARD1 nuclear localization is seen in LNCaP and E006AA-hT cells and increases with androgen treatment. Subcellular distribution of ARD1 proteins was analyzed by confocal fluorescence microscopy. The ARD1 antibody (Santa Cruz Cat# SC-3320) and the Alexa Fluor 488 Anti-rabbit IgG antibody (Cell Signaling Cat# 4412) were used to probe ARD1 in PCa cells. LNCaP and E006AA-hT cells were respectively cultured in phenol red-free RPMI-1640 and phenol red-free DMEM media supplemented with 10% charcoal-stripped FBS. The cells were treated with or without 1 nM R1881 for 24 h and subsequently fixed with 4% paraformaldehyde. The 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. An average of six fields with 10 cells per field were captured for each group. The NIH Image J Software was used to quantify the nuclear (N) and cytoplasmic (C) fluorescent signals. The ratios of nuclear to cytoplasmic (N/C) signals were calculated to demonstrate ARD1 protein nuclear translocation
Fig 2: 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
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