The radiation survived NSCLC cells show upregulation of beta-catenin, Oct-4 and Sox-2 transcription factors. Non-irradiated NSCLC cells, radiation survived adherent cells, non-irradiated sphere cells and radiation survived sphere cells were collected and seeded into collagen precoated 96-well plates. 8 hours later the cells were fixed, permeabelized and immunofluorescently stained for beta-catenin, Oct-4 and Sox-2. The cell nuclei were stained with Hoechst33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (40× objective) and analyzed using the Compartment Analysis BioApplication Software Module. (A) The representative images of parental non-irradiated H460 cells, radiation survived adherent cells, non-irradiated sphere cells and radiation survived sphere cells stained for beta-catenin are shown. (B-D) The total average fluorescence intensities of nuclear beta-catenin (B), Oct-4 (C) and Sox-2 (D) in the non-irradiated NSCLC cells (grey), in the radiation survived adherent cells (green), in the non-irradiated sphere cells (red) and in the radiation survived sphere cells (blue) are presented. Fluorescence intensities of respective IgG controls were subtracted. Each point presents average intensities (pixels) estimated for 3000 cells.