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. 2014 Sep 1;12(7):395–418. doi: 10.1089/adt.2014.594

Fig. 2.

Fig. 2.

TIF2-GFP localization in the nucleolus and AR-TIF2 PPIs. (A) Colocalization of TIF2-GFP with fibrillarin in nucleoli. 40×grayscale and color composite images of U-2 OS cells infected with the TIF2-GFP adenovirus alone, which were cultured overnight and then fixed and stained with a specific antifibrillarin antibody, were sequentially acquired on the ImageXpress Micro (IXM) platform in three fluorescent channels: Ch1 Hoechst, blue; Ch2 TIF2-GFP, green; and Ch3 fibrillarin, red. Representative images from one of the two independent experiments are shown. (B) Control grayscale and color composite images of U-2 OS cells coinfected with both the TIF2-GFP and AR-RFP rAVs. 40× grayscale and color composite images of U-2 OS cells coinfected with both the TIF2-GFP and AR-RFP adenoviruses, cultured overnight and then fixed and stained with Hoechst, were sequentially acquired on the IXM platform in three fluorescent channels: Ch1 Hoechst, blue; Ch2 TIF2-GFP, green; and Ch3 AR-RFP, red. Representative images from one of the two independent experiments are shown. (C) Grayscale and color composite images of U-2 OS cells coinfected with both the TIF2-GFP and AR-RFP rAVs and the treated with DHT. 40× grayscale and color composite images of U-2 OS cells coinfected with both the TIF2-GFP and AR-RFP adenoviruses, cultured overnight, treated±20 nM DHT for 30 min and then fixed and stained with Hoechst, were sequentially acquired on the IXM platform in three fluorescent channels: Ch1 Hoechst, blue; Ch2 TIF2-GFP, green; and Ch3 AR-RFP, red. Representative images from one of the two independent experiments are shown. (D) Pairwise coinfection experiments with the AR-RFP, TIF2-GFP, hDM2-RFP, and p53-GFP adenovirus biosensors. 40× color composite images of U-2 OS cells coinfected with pairwise combinations of the AR-RFP, TIF2-GFP, hDM2-RFP, and p53-GFP adenoviruses, cultured overnight, treated±20 nM DHT or 10 μM Nutlin-3 for 30 min and then fixed and stained with Hoechst, were sequentially acquired on the IXM platform in three fluorescent channels: Ch1 Hoechst, blue; Ch2 TIF2-GFP or p53-GFP, green; and Ch3 AR-RFP or hDM2-RFP, red. The following pairwise combination of the biosensor adenoviruses were tested: AR-RFP plus TIF2-GFP, AR-RFP plus p53-GFP, hDM2-RFP plus TIF2-GFP, and hDM2 plus p53-GFP. Representative images from one of the two independent experiments are shown.