Effect of TRAIL on DDR proteins is observed at all phases of the cell cycle and is not restricted to HCT116 cells. (A) Relationships between γ-H2AX cellular content and cell cycle. Untreated (upper left panel) and TRAIL-treated (0.1 μg/ml, 2 h) HCT116 cells (upper right panel) were analyzed by FACScan flow cytometry. The x axis indicates the DNA content (as determined by propidium iodide staining), and the y axis indicates γ-H2AX content (cells positive for γ-H2AX are in red, and the negative cells are in gray). Results are quantitated in the lower panel. (B) Effects of TRAIL on Chk2, ATM, H2AX, and DNA-PK phosphorylations in HeLa cells. Protein phosphorylations were analyzed by Western blotting. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68). Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated at the bottom. (C) Activation of Chk2 in response to TRAIL in Jurkat cells. Cells were treated with 0.1 μg/ml TRAIL for the indicated times, and the phosphorylation of Chk2 on T68 was analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated.
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