Fig. 6.

Gene regulation by a DNA-binding mutAR. A, Hormone-depleted EP-LNCaP cells were nucleofected with mutAR or control (Ctrl) vector; 24 h later, the cells were infected with AR shRNA lentivirus or nontarget control lentivirus for 48 h. During the last 12 h of the infection, the cells were treated with R1881 (1 nm) or vehicle. The cells were then harvested to measure mRNA levels for the indicated androgen target genes by real-time RT-PCR. B, HeLa cells were depleted of hormone and transfected with the minimal ARE-driven promoter-Luc reporter plasmid and cotransfected with wtAR or mutAR expression plasmid or with the vector control. Testosterone (10 nm) or vehicle was added to the culture media 24 h after transfection; 24 h later, the cells were harvested to measure luciferase activity. C, Hormone-depleted HeLa cells were transfected with the (C/EBP)₃-TATA-Luc construct. The cells were cotransfected with expression plasmid for wtAR, DNA-binding mutAR, or control plasmid as well as C/EBPα expression plasmid or control plasmid. The wtAR and mutAR plasmids were transfected at a dose (200 ng plasmid/1 × 10⁵ cells) at which AR has previously been shown (45) to enter the nucleus in HeLa cells independent of hormone. In addition, all wells were cotransfected with the Renilla luciferase transfection control plasmid; 48 h after transfection, the samples were harvested either to measure luciferase activity or for Western blot analysis. The promoter activity values are plotted as the ratio to the basal activity of the control. Triplicate samples were included in each experimental set. C, inset, Western blotting showing the expression of wtAR, mutAR, C/EBPα, and GAPDH (loading control). D, Hormone-depleted LP50 cells were nucleofected with mutAR or control vector; 24 h later, the cells were infected with AR shRNA lentivirus or nontarget control lentivirus for 48 h. The cells were then harvested to measure mRNA levels for the TMPRSS2 gene by real-time RT-PCR.