FIG. 3.

Analyses of γ-H2AX responses to TRAIL. (A) γ-H2AX confocal immunofluorescence staining in HCT116 cells treated with TRAIL. Images are representative of cells treated with 0.1 μg/ml TRAIL for 1 or 2 h. γ-H2AX was labeled in green, and nuclei were stained in red with propidium iodide (PI). (B) Single-cell analyses showing typical γ-H2AX patterns. From left to right: untreated cell, irradiated cell, and cells treated with TRAIL. The graph shows the relative distribution of the different γ-H2AX patterns. HCT116 cells were treated with 0.1 μg/ml TRAIL for 1 or 2 h. White columns correspond to peripheral nuclear staining (ring pattern, I), gray columns correspond to panstaining (flooded pattern, II), and black columns correspond to apoptotic bodies fully stained with γ-H2AX (III). The distribution of the three γ-H2AX patterns was significantly different for the 1- and 2-h TRAIL treatments (chi-square test, P < 0.05). (C) γ-H2AX confocal immunofluorescence staining in PrEC cells treated with TRAIL (0.1 μg/ml for 6 h). γ-H2AX was labeled in green, and nuclei were stained in red with propidium iodide. (D) Western blotting for γ-H2AX response to TRAIL in PrEC cells. Cells were treated with 0.1 μg/ml TRAIL for the indicated times. HCT116 cells treated with 0.1 μg/ml TRAIL for 2 h were used as a positive control. The percentage of γ-H2AX-positive cells (determined by immunofluorescence microscopy) is indicated for each time point.