Fig. 4.

Titration of the AR-RFP and TIF2-GFP rAV biosensors in U-2 OS cells. (A) Color composite images of U-2 OS cells infected with increasing amounts of the AR-RFP biosensor alone. Increasing volumes (μL) of the AR-RFP rAV biosensor were incubated with 1× 10⁶ U-2 OS cells and 2,500 infected cells were seeded into the wells of 384-well assay plates and cultured overnight at 37°C, 5% CO₂, and 95% humidity. Cells were then fixed and stained with Hoechst 33342 and 10× images of three fluorescent channels (Ch1, blue; Ch2, green; and Ch3, red) were acquired on the IXU automated imaging platform as described previously. Representative color composite images of U-2 OS cells infected with the indicated volumes of AR-RFP virus are presented. (B) Color composite images of U-2 OS cells infected with increasing amounts of the TIF2-GFP biosensor alone. Increasing volumes (μL) of the TIF2-GFP rAV biosensor were incubated with 1×10⁶ U-2 OS cells and 2,500 infected cells were seeded in the wells of 384-well assay plates and cultured overnight at 37°C, 5% CO₂, and 95% humidity. Cells were then fixed and stained with Hoechst 33342 and 10× images of three fluorescent channels (Ch1, blue; Ch2, green; and Ch3, red) were acquired on the IXU automated imaging platform as described previously. Representative color composite images of U-2 OS cells infected with the indicated volumes of TIF2-GFP virus are presented. (C) Average intensity of AR-RFP in the cytoplasm of U-2 OS cells infected with increasing amounts of the AR-RFP biosensor, alone or in combination with the TIF2-GFP biosensor. The multiwavelength translocation image analysis module derived mean±SD (n=8) average cytoplasm (outer) intensity of AR-RFP in U-2 OS cells infected with the indicated volumes of AR-RFP rAVs, either alone (●) or in combination (○) with TIF2-GFP, are presented. Representative experimental data from one of several independent experiments are shown. (D) Average intensity of TIF2-GFP in the nucleoli of U-2 OS cells infected with increasing amounts of the TIF2-GFP biosensor, alone or in combination with the AR-RFP biosensor. The multiwavelength translocation image analysis module derived mean±SD (n=8) average nuclear (inner) intensity of TIF2-GFP in U-2 OS cells infected with the indicated volumes of TIF2-GFP rAVs, either alone (●) or in combination (○) with AR-RFP, are presented. Representative experimental data from one of several independent experiments are shown.