Validation of U2transLUC assay using a panel of test compounds. A. The nuclear accumulation of the GFP-FOXO reporter protein and FOXO driven luciferase activity induced by the test compounds. We exposed U2transLUC cells to three different concentrations of the test compounds. The results shown here are the values obtained from the treatment of U2transLUC cells with 4 nM Ratjadone A, 20 μM LY294002, 1 ng/ml PDGF, 100 μM Minerval, 1 μM Forskolin, 4 nM Leptomycin B, 30 μM Cisplatin, 100 nM PI-103, 0.5 nM Rapamycin, 20 ng/ml IGF, 5 ng/ml EGF and Dimethyl sulfoxide (DMSO) as a negative control. The grey bar graphs and the corresponding left hand y-axis depict the values in relation to the control values normalized to the corresponding measure of viability values. The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the average GFP intensity from the same well. The right hand y-axis and the hatched bars indicate the percentage of cells in each well exhibiting nuclear/cytoplasmic (Nuc/Cyt) ratios of fluorescence intensity greater than 1.8 normalized to the percentage in DMSO-treated wells. B. Representative images of treated cells using high throughput format of the U2transLUC system. Images of cells expressing GFP-FOXO and stained with DRAQ5 were obtained by automated microscopy 2 hour after drug exposure. The images correspond to U2transLUC cells exposed to 4 nM Ratjadone A, 4 nM Leptomycin B, 20 μM LY294002, 100 nM PI-103 and DMSO. The images are shown from the GFP and DRAQ5 channels, as well as the corresponding merged image in the case of the DMSO treated control cells.