Fig. 3.

Translocation enhanced (TE) image analysis module. (A) Image segmentation derived TIF2-GFP-positive nucleoli masks in the FITC and Texas Red channels. Enlarged and cropped grayscale images of TIF2-GFP (Ch2) and AR-RFP (Ch3) from U-2 OS cells coinfected with both the TIF2-GFP and AR-RFP adenoviruses, cultured overnight and then treated±20 nM DHT for 30 min. The TE image analysis module utilized the TIF2-GFP biosensor component in Ch2 to create a mask of the nucleoli. The TIF2-GFP was localized to bright fluorescent puncta within the Hoechst-stained nuclei of U-2 OS cells and objects in Ch2 with TIF2-GFP fluorescent intensities >750 gray levels over background, with an approximate width of 4.0 μm, a minimum area of 5.0 μm², and that did not exceed a maximum area of 150 μm² were classified by the image segmentation as TIF2-GFP-positive nucleoli and used to create translocation masks within the nucleus of cells. AR-RFP images from Ch3 were segmented into a nucleolus region using the mask derived from the detected TIF2-GFP-positive nucleoli in Ch2. The red or green color of the nucleolus masks indicate where the correlation coefficient between the TIF2-GFP and AR-RFP signals within the nucleoli were below (red) or above (green) a preset threshold (typically≥0.25). Quantitative data extracted by the TE image analysis module: (B) Number of the TIF2-GFP-positive compartments (selected objects) or nucleoli count in Ch2; (C) average fluorescent intensity of the TIF2-GFP-stained nucleoli in Ch2; (D) average fluorescent intensity of the AR-RFP signal in Ch3 within the TIF2-GFP-positive defined nucleolus region; and (E) integrated fluorescent intensity of the AR-RFP signal in Ch3 within the TIF2-GFP-positive defined nucleolus region. The enlarged grayscale images and the derived masks of nucleoli are representative of those obtained in numerous independent experiments. The quantitative data represent the mean±standard deviation (SD) (n=32) of 32 DMSO-treated (0.2%) and DHT-treated (20 nM in 0.2% DMSO) wells from one of numerous independent experiments. The mean average inner intensity of AR-RFP within the TIF2-GFP-positive nucleoli mask of Ch3 (D) was selected as the primary output of the TE image analysis module to quantify DHT-induced AR-TIF2 PPIs.