The subcellular localization of Bach2 is regulated by the PI-3K/S6K pathway. Representative images of Bach2 (left panels), DNA (middle panels), and merged images (right panels) are shown for each experiment. (A) BV173 cells were either exposed or not (control) to 10 μM imatinib (IM) for 24 hours. (B) Namalwa cells were untreated (control) or treated with 100 μM LY294002 (LY) or 1 μM wortmannin for 2 hours. (A-B) Cells were fixed and immunostained with anti-Bach2 polyclonal antibody and Hoechst 33342, the coverslips were mounted in DAPI-containing Vectashield (Vector Labs, Burlingame, CA), and the slides were observed under a 40×/1.0 NA oil objective through a Zeiss LSM510 confocal laser scanning microscope. (C) 3T3 cells were transiently transfected with pEGFPBach2 or pEGFPBach2S521A. The coverslips were mounted in DAPI-containing Vectashield, and the slides were examined under a 40×/1.0 NA oil objective through an Olympus BX-41 fluorescence microscope (Olympus, Tokyo, Japan). Images were captured using an Orca AG digital camera (Hamamatsu Photonics UK, Hertfordshire, United Kingdom) and Smartcapture X software (Digital Scientific, Cambridge, United Kingdom) and were processed with Adobe Photoshop CS (Adobe Systems, San Jose, CA).