ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 2. Down-regulation of Bmi-1 causes defective proliferation in Atm-/- NSCs. (A) Generation of Bmi-1 over-expressing NSCs by infecting Atm+/+ or Atm-/- NSCs with MSCV-GFP-bmi-1 bearing retrovirus. (B) Neurosphere number generated by control and Bmi-1 over-expressing Atm+/+ and Atm-/- NSCs. (C) Total cell number per well generated by control and Bmi-1 over-expressing Atm+/+ and Atm-/- NSCs. (D) Neurosphere size generated by control and Bmi-1 over-expressing Atm+/+ and Atm-/- NSCs. (B-D) The mean6S.E. of three independent experiments is shown. Np,0.05; NNp,0.01, when GFP-Bmi-1 expressing Atm+/+ NSCs were compared with GFP expressing Atm+/+ NSCs, when GFP-Bmi-1 expressing Atm-/- NSCs were compared with GFP expressing Atm-/- NSCs, or when GFP expressing Atm-/- NSCs were compared with GFP expressing Atm+/+ NSCs. (E) Phase-contrast photomicrographs of Atm+/+ and Atm-/- neurospheres, either expressing MSCV-GFP or MSCV-GFP-Bmi-1. Scale bars = 50 mm. doi:10.1371/journal.pone.0016615.g002 evident in floating neurospheres, as shown by increased neuro- sphere number and size in Atm-/- NSCs. Collectively, these results demonstrate that NSCs require Bmi-1 for survival and normal proliferation, and that down-regulation of Bmi-1 substantially contributes to the defective proliferation of Atm-/- NSCs. Bmi-1 undergoes proteasome-mediated degradation, which is accelerated during oxidative stress Other researchers demonstrated that Bmi-1 expression is modulated through transcriptional and posttranscriptional regula- tion in hematopoietic stem cells [15]. Here, we observed no distinct differences in bmi-1 mRNA levels between Atm+/+ and Atm-/- NSCs, although decreased level of Bmi-1 protein was noted in Atm-/- NSCs (Figure 1). These results indicate that Bmi-1 may be downregulated via a posttranscriptional mechanism in Atm-/- NSCs. To determine if the level of Bmi-1 is regulated via posttranscriptional proteasome- mediated degradation in NSCs, we examined Bmi-1 stability in Atm+/+ NSCs treated with the protein synthesis inhibitor cyclohex- imide. Cycloheximide-treated cells showed a rapid decline in Bmi-1 levels with half-life about 2 hours (Figure 3A). To investigate whether proteasome inhibition affects the levels of endogenous Bmi- 1, NSCs were treated with proteasome inhibitor MG132. Treatment of cells with MG-132 significantly increased Bmi-1 levels compared to those in untreated cells (Figure 3B). In addition, co-treatment with MG-132 and cycloheximide slowed the degra- dation of Bmi-1, compared to single treatment with cycloheximide (Figure 3C). These results indicate that Bmi-1 turn over can be antagonized by proteasome inhibition. We have previously demonstrated that excess ROS elicited by H2O2 treatment decreased NSC proliferation through activation of p38 signaling, similar to that in Atm-/- NSCs [6]. Therefore, to ascertain whether oxidative stress could also affect Bmi-1 degradation, we treated Atm+/+ NSCs with H2O2 and analyzed Bmi-1 and p21 levels. Western blot analysis revealed that Bmi-1 appears as a triplet of approximately 40 to 44-kDa. Currently, no specific antibody is available to detect the phosphorylated form of Bmi-1, thus phosphorylation is determined by mobility shift. After 1 hour of treatment with H2O2, the unphosphorylated Bmi-1 band was significantly reduced (faster migration on gel), resulting in an increase in the phosphorylated bands (slower migration on gel), compared to those of untreated samples. After 4 hours of incubation with H2O2, we observed a dramatic reduction in the PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16615