Fig. 8.

Concentration-dependent confirmation of LOPAC actives that inhibit the DHT-induced formation of AR-TIF2 PPIs or disrupt preexisting AR-TIF2 PPI complexes. (A) Concentration-dependent inhibition of DHT-induced AR-TIF2 PPI formation by NR ligands. U-2 OS cells were coinfected with the AR-RFP and TIF2-GFP rAV biosensors, 2,500 cells were seeded into the wells of 384-well assay plates, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and then exposed to compounds at the indicated concentrations for 3 h. Cells were then treated with 20 nM DHT for 30 min, fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=3) percent inhibition of DHT-induced AR-TIF2 PPIs in cells exposed to the indicated concentrations 17-alpha-hydroxyprogesterone (17-α-H-PG) (○), nilutamide (■), 2-methoxyestradiol (2-MOED) (▼), or estrone (▽) are presented. Representative experimental data from one of the three independent experiments are shown. (B) Concentration-dependent inhibition of DHT-induced AR-TIF2 PPI formation by non-NR ligands. U-2 OS cells were coinfected with the AR-RFP and TIF2-GFP rAV biosensors, 2,500 cells were seeded into the wells of 384-well assay plates, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and then exposed to compounds at the indicated concentrations for 3 h. Cells were then treated with 20 nM DHT for 30 min, fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=3) percent inhibition of DHT-induced AR-TIF2 PPIs in cells exposed to 4-phenyl-3-furoxancarbonitrile (4-P-3-FOCN) (●), Bay 11-7085 (■), parthenolide (Δ), 1-chloro-3-tosylamino-4-phenyl-2-butanone (TPCK) (▼), or N-Carbobenzyloxy-L-phenylalanyl chloromethyl ketone (ZPCK) (○) are presented. Representative experimental data from one of the three independent experiments are shown. (C) Concentration-dependent confirmation of disruptor actives identified in the AR-TIF2 PPIB LOPAC screens. U-2 OS cells were coinfected with the AR-RFP and TIF2-GFP rAV biosensors, 2,500 cells were seeded into the wells of 384-well assay plates, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and then exposed to 20 nM DHT for 30 min. Cells were then treated with compounds at the indicated concentrations for 3 h fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=3) percent disruption of preformed AR-TIF2 PPI complexes in cells exposed to the indicated concentrations of mifepristone (●), 4-phenyl-3-fluroxane carbo-nitrile (4-P-3-FOCN) (□), and gugglesterone (■) are presented. Representative experimental data from one of the three independent experiments are shown.