p38 MAPK-Mediated Bmi-1 Down-Regulation and Defective Proliferation in ATM-Deficient Neural Stem Cells Can Be Restored by Akt Activation
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 µg/mL) and Bmi-1 levels were determined by Western blotting. Probing with an antibody to β-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 β-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 µg/mL) and MG132 (10 nM), and Bmi-1 levels were determined by Western blotting. Probing with an antibody to β-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 µM 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 µM 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 µg/mL) and 100 µM H2O2, and Bmi-1 levels were determined by Western blotting. Probing with an antibody to β-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 µM SB203580 for 2 hours before treated for the times indicated with cycloheximide (50 µg/mL), and Bmi-1 levels were determined by Western blotting. Probing with an antibody to β-actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point.