FIG.4.

Inhibition of BAF57 results in reduced endogenous AR activity. (A) LNCaP cells (2 × 10⁵) were transfected with 1 μg of pARR2-Luc reporter, 0.25 μg of pTK-Renilla luciferase, and either 2.25 μg of pBabe-BAF57ΔN (dominant-negative BAF57) or parental vector (to 4.5 μg of total DNA). Cells were treated with either 0.1% EtOH vehicle or 1 nM DHT for 48 h (with restimulation after 24 h) and then harvested and lysed. The luciferase activity was measured as in Fig. 3D, and the AR activity in the presence of vector alone plus DHT was set to 100. The average relative luciferase activity and standard deviations are shown. ✽, P < 0.05. (B) LNCaP cells transfected with either vector (lane 1) or BAF57ΔN (lane 2) were harvested, and the expression of endogenous AR and CDK4 (loading control) was determined by immunoblotting. (C) LNCaP cells (4.5 × 10⁵) were transfected with 1 μg of pBabe-PURO, 1 μg of H2B-GFP, and 4 μg of parental vector (lane 1), BAF57ΔN (lane 2), or dominant-negative AR (dnAR) (lane 3) expression plasmids. Posttransfection, cells were selected with puromycin until >90% of cells were H2B-GFP positive (ca. 3 days). Cells were then harvested, and RNA was isolated and subjected to RT-PCR. cDNAs were amplified in the presence of [³²P]dCTP to accurately quantify the signal (upper panel). Product levels were quantified by using a PhosphorImager. Relative levels of PSA over GAPDH from two independent experiments, and standard deviations are shown (lower panel).