Figure 7

Regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression by GSK3β. A: The GSK3β inhibitor LiCl prolongs phosphorylated H2AX increase in response to 4 Gy irradiation. HeLa cells were pretreated with 40 μM LiCl for 2 h, irradiated with 4 Gy γ rays and harvested at 0, 0.5, 1, 4 and 10 h after irradiation. Protein expression was assayed by Western blotting. B: GSK3β inhibitor LiCl enhanced the phosphorylation of H2AX in G2/M phase cells. To release the cells from G1 block and inhibit GSK3β activity, synchronized HeLa cells were grown in DMEM medium supplemented with 40 μM LiCl. S-phase cells were harvested at 5 h and G2/M phase cells at 8, 9 and10 h after released. Protein expression was assayed by Western blotting. C: RNAi depletion of GSK3β. HeLa cells were transfected with 50 nM GSK3β siRNA or non-specific (ns) control siRNA molecules. GSK3β expression was determined by Western blotting. D: Effect of GSK3β depletion on the phosphorylation of H2AX induced by 4 Gy γ-irradiation. After 48 h incubation with 50 nM GSK3β-specific siRNA or control non-specific (ns), cells were irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting. E: Effect of the PP2A inhibitor fostriecin on H2AX phosphorylation. HeLa cells were pretreated with 50 nM fostriecin for 2 h, and then irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting.