Fig. 5.

AR-TIF2 PPIB assay development. (A) DHT activation time course. 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 treated with 20 nM DHT for the indicated time periods. Cells were then fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the DHT-induced AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=16) (●) average inner intensity of AR-RFP within the TIF2-GFP-positive nucleoli at time 0 and various time points ranging from 10 to 120 min are presented. Representative experimental data from one of the three independent experiments are shown. (B) DHT concentration responses. 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 treated with the indicated concentrations of DHT for 30 min. Cells were then fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the DHT-induced AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=3) average inner intensity of AR-RFP within the TIF2-GFP-positive nucleoli at concentrations ranging between 0.001 and 100 nM DHT are presented. Representative experimental data from four independent experiments are shown: experiment 1 (●), experiment 2 (○), experiment 3 (■), and experiment 4 (□). (C) DMSO tolerance. 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, cells were exposed to the indicated DMSO concentrations for 1 h, and then treated±20 nM DHT for 30 min. Cells were then fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the DHT-induced AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=4) average inner intensity of AR-RFP within the TIF2-GFP-positive nucleoli in control (○) and 20 nM DHT-treated (●) cells at DMSO concentrations ranging between 0.001% and 10% DMSO are presented. Representative experimental data from one of the three independent experiments are shown. (D) U-2 OS cell seeding density. U-2 OS cells were coinfected with the AR-RFP and TIF2-GFP rAV biosensors and then seeded into the wells of 384-well assay plates at the indicated cell densities, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and then treated±20 nM DHT for 30 min. Cells were then fixed and stained with Hoechst, 20× images in three fluorescent channels were acquired on the IXU automated imaging platform, and the DHT-induced AR-TIF2 PPIs were quantified using the TE image analysis module as described previously. The mean±SD (n=6) average inner intensity of AR-RFP within the TIF2-GFP-positive nucleoli in DMSO control (○) and 20 nM DHT-treated (●) cells at seeding densities ranging between 625 and 20,000 cells per well are presented. Representative experimental data from one of the three independent experiments are shown.