Figure 2. TC-AR is transcriptionally active in the absence of DHT and confers ADI growth in vitro.

A Luciferase assay showing DHT-independent transcription of a transiently transfected AR regulated promoter following induction of TC-AR in LN/TC-AR. LN/ TC-AR cells were co-transfected with pPSA6.0-luc and pH 48-ren in hormone depleted media and treated with either low concentrations of doxycycline, DHT 1 nM or vehicle as control for 24 hours. Fold induction is relative to untreated control. B Immunostaining of LN/TC-AR shows androgen independent nuclear localization of TC-AR (right). Cells were counterstained with DAPI to identify nuclei (left) and images were acquired with an Olympus fluorescent microscope using 20× magnification. C Chromatin Immunoprecipitation (ChIP) showed the recruitment of AR and RNA polymerase II to the KLK3 promoter. LN/TC-AR cells were pre-cultured in androgen depleted medium for 72 hours then treated with Low Dox for 24 hours. Anti-FLAG M2 (for FLAG-tagged TC-AR) and α-RNAP2 antibody were used in separate aliquots to immunoprecipitate cross-linked protein and DNA. D Cell count assay showing the growth of LN/TC-AR in hormone depleted media following treatment with 1 nM DHT, Low Dox, High Dox or vehicle as control.