ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 3. Bmi-1 undergoes proteasome-mediated degradation, which is accelerated during oxidative stress. (A) Atm+/+ NSCs were treated for the times indicated with cycloheximide (50 mg/mL) and Bmi-1 levels were determined by Western blotting. Probing with an antibody to b- actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. (B) Atm+/+ NSCs were treated for the times indicated with MG132 (10 nM) and Bmi-1 levels were determined by Western blotting. Probing with an antibody to b-actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. (C) Atm+/+ NSCs were co-treated for the times indicated with cycloheximide (50 mg/mL) and MG132 (10 nM), and Bmi-1 levels were determined by Western blotting. Probing with an antibody to b- actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. (D) NSCs from Atm+/+ mice were treated for the times indicated with 100 mM H2O2 with the addition of MG132 (10 nM), and Bmi-1 and p21 levels were determined by Western blotting. (E) NSCs from Atm+/+ or Atm-/- mice were treated for 7 hours with 100 mM H2O2 and total RNA was purified and analyzed by quantitative RT- PCR analysis for bmi-1, p21, p16, and p19 expression. Probing for gapdh was used as an internal control. (F) NSCs from Atm+/+ mice were treated for the times indicated with cycloheximide (50 mg/mL) and 100 mM H2O2, and Bmi-1 levels were determined by Western blotting. Probing with an antibody to b-actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. (G) NSCs from Atm+/+ mice were pretreated with 10 mM SB203580 for 2 hours before treated for the times indicated with cycloheximide (50 mg/mL), and Bmi-1 levels were determined by Western blotting. Probing with an antibody to b-actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. doi:10.1371/journal.pone.0016615.g003 level of phosphorylated Bmi-1 (decreased by 90% compared to the untreated sample), as well as a significant reduction in the level of Bmi-1 (Figure 3D). In addition, H2O2 caused a significant increase in p21 protein levels in NSCs. Interestingly, H2O2 selectively caused a significant elevation in p21 expression, whereas expression of bmi-1, p16, and p19 were not altered in H2O2- treated NSCs (Figure 3 E). The reason of the selectivity of p21 expression by oxidative stress is unclear. However, it can be explained by the previous study demonstrating that p21 protects cells from oxidative damage through activation of Nrf2 as an antioxidant response [16], and it was also shown that Bmi-1 is important for NSC proliferation and survival at earlier embryonic stages, via suppression of cell cycle inhibitor p21, potentially with stage-related differences [11]. The result as shown above that Bmi-1 is posttranscriptionally downregulated in response to H2O2 (Figure 3D and E) prompted us to examine whether oxidative stress causes a change in Bmi-1 turnover. Wild-type NSCs were co-treated with H2O2 and cycloheximide. We observed that H2O2 dramatically shortens Bmi-1 half-life (Figure 3F), whereas the p38 inhibitor SB203580 slowed this rapid degradation of Bmi-1 (Figure 3G), suggesting that the H2O2-induced rapid degradation of Bmi-1 is a p38- dependent pathway. This finding is consistent with our previous observation that oxidative stress reduces proliferation of NSCs through p38 signaling [6]. Collectively, these results demonstrate that Bmi-1 undergoes proteasome-mediated degradation, and that oxidative stress elicited by H2O2 accelerates Bmi-1 degradation through p38 signaling in NSCs. PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16615