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. 2016 Jun 3;7(26):40690–40703. doi: 10.18632/oncotarget.9816

Figure 2. Androgen depletion accentuates enzalutamide agonism of mutant F877L AR.

Figure 2

A. Trypan blue cell viability assay of enzalutamide-resistant MR49F cells cultured with charcoal-stripped serum. Cells were grown for four days in medium containing charcoal-stripped serum, and then cells were treated with fresh medium containing either vehicle, 10 μM enzalutamide, 1 nM DHT, or the combination for six days. Treatment media was changed on day three. Data are means of three biological replicates; error bars represent standard deviations. * = p≤0.05, ** = p≤0.01, unpaired 2-tailed t-test. Comparisons are to vehicle treatment. B. RT-qPCR was used to quantify mRNA expression of canonical AR targets KLK3, TMPRSS2, and NKX3.1 in MR49F cells cultured with charcoal-stripped serum. Cells were grown for three days in medium containing charcoal-stripped serum, and then cells were treated with fresh medium containing either vehicle, 10 μM enzalutamide, 1 nM DHT, or the combination for 24 hours. Data are mean RQ (ΔΔCt method) of three biological replicates; positive and negative error bars represent standard error of the mean (SEM). ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001, unpaired 2-tailed t-test. Comparisons are to vehicle treatment. C. Western blots of protein lysates from experiments above in (B) were used to measure PSA protein expression in MR49F cells. D. LNCaP cells stably overexpressing ectopic F877L AR and a probasin promoter GFP reporter (LNCaP Pb. EGFP ARF877L) were grown in full serum or charcoal-stripped serum and then treated with fresh medium containing either vehicle, 1 nM DHT, or 10 μM enzalutamide for six days. Treatment media was changed on day three. For the charcoal-stripped condition, cells were switched to medium with charcoal-stripped serum for 24 hours prior to drug treatment. GFP expression was measured with flow cytometry. Data are means of three biological replicates; error bars represent SEM. **** = p≤.0001, unpaired 2-tailed t-test. E. MR49F cells were grown in medium containing charcoal-stripped serum for three days, and then treated with fresh medium containing either vehicle, 1 nM DHT or 10 μM enzalutamide for 24 hours prior to harvest for RNA-seq. Venn diagram of RNA-seq data demonstrating overlap of genes induced by DHT or enzalutamide treatment. Expression data per gene represents the mean read count values of three biological replicates. After variance stabilizing the data using DESeq, we used a t-test to determine significant differentially-expressed genes in the drug-treated vs. vehicle-treated conditions (FDR-adjusted p-value ≤0.05).