Figure 3. BET bromodomain inhibition prevents activation of mutant F877L AR and suppresses CRPC cell viability in androgen-depleted conditions.

A. MTS cell viability assay of MR49F cells. Cells were grown for three days in charcoal-stripped serum, and then we treated cells with enzalutamide (10 μM) and DHT (1 nM) or vehicle +/− 500 nM JQ-1 for three days. Data are the means of six biological replicates; error bars represent standard deviations. **** = p≤.0001, unpaired 2-tailed t-test. B. RT-qPCR to measure mRNA expression of AR target genes in MR49F cells from experiments described in (A). Data are mean RQ (ΔΔCt method) of three biological replicates; positive and negative error bars represent SEM. ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001, unpaired 2-tailed t-test. Comparisons are between respective vehicle and JQ-1 treated groups (ENZ+ vehicle vs. ENZ+JQ-1, etc.). C. Western blot was used to measure PSA protein expression in MR49F cells treated as in (A). D. Heat map of RNA-seq gene expression changes in MR49F cells treated as in (A). 57 shared enzalutamide and DHT-regulated genes from Figure 2E whose expression is significantly reversed (≥2 fold) by JQ-1 are shown. Expression data per gene represent the mean read count value of three biological replicates. After variance stabilizing the data using DESeq, we used a t-test to determine significant differentially-expressed genes in the JQ-1-treated conditions (average of enzalutamide + JQ-1 and DHT + JQ-1) when compared to vehicle (average of enzalutamide+vehicle and DHT+vehicle). FDR-adjusted p-value ≤0.05. E. Luciferase reporter assay of AR function in AR-null PC3 cells transfected with ectopic mutant F877L AR. Cells were cultured in charcoal-stripped serum and transfected with an ectopic AR F877L plasmid, an ARE-4 firefly luciferase reporter plasmid, and a Renilla luciferase control plasmid for 72 hours. Data are means of three biological triplicates; error bars represent standard deviation. **** = p≤0.0001, unpaired 2-tailed t-test.