Figure 6. AR negatively regulates BMX gene transcription.

(A) VCaP cells in steroid depleted medium were treated with DHT, without or with 10 μM of Bicalutamide (Bic) for 24 hr and BMX mRNA was measured using qRT-PCR. (B) VCaP cells were cultured in Charcoal-Stripped Serum (CSS) containing medium (androgen depleted) with or without 10 nM DHT for 72 hr (Left panel). VCaP cells in CSS medium for 48 h were treated with vehicle, DHT, or enzalutamide (ENZ, 10μM) for 24 hr. (C) BMX and BTK protein expression in a series of PCa cell lines (LNCaP-BMX cells are stably transfected with BMX). (D) AR binding sites in BMX loci after 4 hr DHT stimulation identified by AR ChIP-seq. (E) VCaP cells in CSS medium were treated with or without 10 nM DHT for 4 hrs followed by CHIP with the indicated antibodies and qPCR for the ABS1 (left) and ABS2 (right) sites (means ± SD of at least 3 biological replicates). (F) VCaP cells were treated with LSD1 inhibitor S2101 (100 μM) for 4 hours with/out 10 nM DHT, and effects on BMX mRNA were analyzed by qRT-PCR (means ± SD, n=3). (G)VCaP cells were treated with or without 10 nM DHT for 4 hrs followed by CHiP-qPCR for FOXA1, p300 or OCT1 binding to ABS1 (left) and ABS2 (right) sites (means ± SD of at least 3 biological repeats).