Figure 6

Regulation of phosphoinositide-dependent kinase (PDK) on the phosphorylation of H2AX. A: RNAi mediated depletion of PDK protein. HeLa cells were transfected with 50 nM PDK specific siRNA molecules or non-specific (ns) control siRNA. Western blotting shows PDK expression. B: Phosphorylation of Akt at Ser473 and GSK3β at Ser9 was decreased in the DNA-PKcs depleted HeLa-H1 cells compared to control HeLa-NC cells. C: PDK regulates the phosphorylation of H2AX in response to DNA damage induced by 4 Gy of γ-irradiation. After 48 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were irradiated with 4 Gy γ rays, then harvested 0, 0.5, 1, 4, 10 h post-irradiation and analyzed by Western blotting. D: PDK regulates the phosphorylation of H2AX associated with cell cycle progression. After 24 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were synchronized in G1 phase by TdR double-blocking, then released and harvested after 5 h, for S-phase, and at 8, 9, and 10 h, for G2/M phase. The culture medium was supplemented with 50 nM siRNA molecules during the period of synchronization and cell cycle progression. Protein expression was assayed by Western blotting.