Figure.2. Agonist-liganded AR negatively regulates AR gene transcription.

(A) VCaP or LNCaP cells were treated with 0, 0.01, 0.1, 1, or 10 nM DHT for 4h, 8h, or 24h and AR mRNA was measured using qRTPCR. (B) VCaP cells were DHT stimulated for 24h and mRNA for PSA and ERG were measured by qRT-PCR. (C) VCaP cells were treated with cycloheximide (10 ng/mL) and DHT or vehicle, and AR mRNA was then measured by qRT-PCR after.0, 1, 4, 8, or 24h (mRNA expression was normalized to internal control 18S RNA in all the experiments). (D) VCaP cells were treated with 0, 0.1, 1, or 10 nM DHT and with 0, 10, or 40 μM bicalutamide for 24h and AR mRNA was measured by qRT-PCR. (E) Left panel - androgen starved VCaP cells were pretreated with DHT or vehicle for 2 hours followed by addition of actinomycin D (10 μM); right panel - VCaP cells growing in medium with DHT were switched to the same medium with or without DHT for 16 h, followed by addition of actinomycin D. AR mRNA was measured by qRT-PCR at the indicated times after actinomycin D addition. Levels at time 0 were normalized to 1 under both conditions in the left panel and under the DHT removal condition in the right panel. Dotted lines indicate 50% maximal level. (F) VCaP cells were treated with/out DHT for 4h. The DNA bound to RNA polymerase II or active RNA polymerase II (phospho-Ser5) was immunoprecipitated and measured by qPCR. Error bars in each experiment indicate standard deviation (SD).