Fig. 2

PDEF is directly regulated by AR. a AR and PDEF mRNA and protein levels were determined by performing RT-qPCR (top left panels) and western blotting (top right panels), respectively, of MDA-MB-453 and SKBR-3 cells treated with 1 nM DHT for 48 h (+DHT) or without DHT (control). AR and PDEF mRNA and protein levels were determined by performing RT-qPCR (bottom left panels) or western blotting (bottom right panels), respectively, of MDA-MB-453 and SKBR-3 cells infected with a non-specific (NS) shRNA- or AR-shRNA-expressing lentiviral vector (KD: knockdown). Data are presented as mean with SD. b AR and PDEF mRNA levels were determined by performing RT-qPCR of MDA-MB-453 (above) and SKBR-3 (under) cells treated with increasing DHT doses for 48 h. The mRNA levels are presented as mean with SD and have been normalised using those of the housekeeping gene GAPDH. c AR and PDEF protein levels were determined by performing immunofluorescence staining of MDA-MB-453 and SKBR-3 cells treated with 1 nM DHT for 0 h (control) or 48 h (+DHT). AR and PDEF protein levels were determined by performing immunofluorescence staining of MDA-MB-453 and SKBR-3 cells infected with an NS shRNA- or AR-shRNA-expressing lentiviral vector (KD: knockdown). d Co-IP assay was performed with an anti-AR antibody in MDA-MB-453 cells treated with 1 nM DHT for 48 h and control vector-infected cells. The interaction between precipitated PDEF and AR was detected using the anti-AR antibody. e Top panel: Schematic diagram of the AR-binding regions within the PDEF locus. Enh: enhancer. Lower left panel: Results of the direct AR ChIP assay followed by RT-qPCR of MDA-MB-453 cells treated with vehicle (white bars) or 1 nM DHT (48 h, black bars); data are presented as mean ± SD. Lower right panel: Semi-quantitative PCR of a negative control (IgG) sample