Fig. 3.

Inhibition of DHT-induced AR-TIF2 PPI responses and corresponding AR-TIF2 distribution phenotypes. U-2 OS cells were co-infected with the AR-RFP and TIF2-GFP rAV biosensors, 2500 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 1 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 above. The mean ± sd (n = 3) average inner intensity of AR-RFP within the TIF2-GFP positive nucleoli in cells exposed to the indicated concentrations of Bicalutamide (●), Flutamide (●), or 17-AAG (●), for 1 h and then treated with 20 nM DHT (●) or 0.5% DMSO (●) are presented. Experimental data from one of five independent experiments are presented. Representative 40× color composite images of the AR-TIF2 biosensor phenotypes of co-infected U-2 OS cells pre-exposed to 0.5% DMSO, 50 μM bicalutamide in 0.5% DMSO, 50 μM flutamide in 0.5% DMSO, or 5 μM 17-AAG in 0.5% DMSO for 1 h prior to 30 min treatment ± 20 nM DHT are shown