Inhibition of NF-κB signaling and nuclear translocation of FoxO3a by GSI I. (A) Western blot of cytoplasmic (C) and nuclear (N) extracts of MyLa cells treated with GSI I (0-5μM, 6 hours) showing a concentration-dependent decrease in the nuclear NF-κB content. The positive control was treated with the NF-κB inhibitor, MG132 (10μM). Immunoblotting with anti-PARP documented the purity of the cytoplasmic and nuclear extracts. (B) The cytoplasmic (C) and nuclear (N) fractions of MyLa cells were obtained as in Figure 4A, and the blots were probed with the FoxO3a-specific antibody. The blot shows a translocation of FoxO3a from the cytoplasm to the nucleus. PARP is a marker of fraction purity, as in Figure 4A. (C) MyLa cells were treated with 5μM GSI I or the vehicle for 4 hours or 12 hours, fixed and stained with the anti-FoxO3a antibody followed by the AlexaFluor568-conjugated secondary antibody. The cells were observed in confocal microscope (Olympus, FluoView Confocal System). Original scale of the images is 50 μm × 50 μm. (D) Nuclear FoxO3a fluorescence was quantified from the confocal microscopy images of CTCL lines treated with GSI I as in panel A (n = 10 cells per group). *P < .05 compared with the control (t test). Bars represent mean ± SD. (E) Phosphorylation of FoxO3a in CTCL cells treated with the vehicle or 5μM GSI I for 4 hours. The cellular content of the whole FoxO3a and FoxO3a phosphorylated at Ser318/321 was determined by Western blot. Compared with MyLa and HuT-78, SeAx had low basal levels of phosohorylated FoxO3a, and band intensity quantification exhibited no decrease in phosphorylation after treatment.