The fraction of cells with cytoplasmic retention of Bach2 varies in response to PI-3K/S6K signaling and oxidative stress. 3T3 cells were transiently transfected with pEGFPBach2 plasmids, and the subcellular localization of GFP-Bach2 was observed through a fluorescence microscope. (A) Percentage of BCR-ABL–positive (+) or –negative (−) 3T3 cells with cytoplasmic GFP-Bach2 when exposed to 0.5 μM imatinib (+IM) for 24 hours, as indicated, and to 300 μM DEM (+DEM) or not (-DEM) for the last 3 hours before fixation. (B) Percentage of BCR-ABL–positive (+) or –negative (−) 3T3 cells with cytoplasmic GFP-Bach2 exposed to 25 μM LY294002 (+LY) or DMSO (-LY) for 2 hours. (C-D) Percentage of 3T3 cells expressing HAS6K1E389D3E, S6K2T401D, or vehicle (pRK7 or pcDNA3.1) with cytoplasmic GFP-Bach2 when treated with 5 μM LY294002 (+LY) or DMSO (-LY) for 2 hours (C) or treated (+DEM) or not (-DEM) with 300 μM DEM for 3 hours (D). (E) Percentage of cells stably expressing Bcr-Abl (+) or vehicle (-) with cytoplasmic GFP-Bach2 (WT) or GFP-Bach2S521A (S521A) treated or not with 300 μM DEM (+DEM) for 3 hours before fixation. At least 100 cells expressing GFP-Bach2 were counted in each experiment. Results are average ± standard deviation of 3 independent experiments. Comparison between the indicated groups shows a significant difference (*) or no significant difference (NS) by the Mann-Whitney U test (P < .05).