ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 4. EGF-mediated Akt signaling causes Bmi-1 phosphorylation and up-regulation in NSCs. (A) Atm+/+ NSCs were deprived of epidermal growth factor (EGF, 20 ng/ml) for 4 hours, at which time EGF was added back to the cultures for indicated times, with or without addition of LY2949002 (LY, 10 mM), and analyzed by Western blotting for phospho-Akt and Akt levels. (B) After EGF starvation of Atm+/+ NSCs for 4 hours, EGF was added back to the cultures for 3 hours. Neurospheres were analyzed by Immunocytochemistry for the levels of ubiqutinated-histone H2A (H2A- Ub) and phospho-Akt. Cells were counterstained by DAPI (49-6-Diamidino-2-phenylindole), which identifies the nuclei of the NSCs. (C) After EGF starvation of Atm+/+ NSCs for 4 hours, EGF was added back to the cultures for 3 hours and analyzed for levels of phospho-Akt, Akt, and Bmi-1 by Western blotting. Protein samples (30 mg) were then treated in vitro with l phosphatase (Ser/Thr and Tyr phosphatase) for 1.5 hours and analyzed for changes in migration of Bmi-1 in SDS-PAGE. Arrowhead indicates totally dephosphorylated forms of Bmi-1 that can be used as the reference band to visualize the mobility shift of Bmi-1 (faster migration on gel). (D) Atm+/+ NSCs and GSCs were deprived of EGF for 4 hours. EGF was added back to the cultures for 3 hours, with or without LY2949002. Bm-1 levels and localization were analyzed by Immunofluorescence (FITC-green). Cells were counterstained by DAPI (49-6-Diamidino-2-phenylindole), which identifies the nuclei of the NSCs. doi:10.1371/journal.pone.0016615.g004 the status of ATM, although total levels of Bmi-1 decreased in Atm-/- NSCs (Figure 6A). This phenomenon might be explained by the fact that under normal conditions Akt is primarily responsible for phosphorylating Bmi-1, but in Atm-/- NSCs, p38 is still capable of phosphorylating Bmi-1 in an ATM-independent manner. Next, we sought to determine whether up-regulation of Bmi-1 and Akt signaling correlated with inhibition of Bmi-1 proteasomal degradation. We treated Atm+/+ and Atm-/- NSCs with MG132 for 4 hours to monitor Akt activation and Bmi-1 levels. We found that MG132 treatment increased Akt activation and Bmi-1 levels in both cells, and that Akt activation by MG132 treatment was accompanied by increased Bmi-1 levels. In addition, the levels of Akt activation and Bmi-1 expression in Atm-/- NSCs treated with MG132 were comparable to levels in MG132-treated Atm+/+ PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16615