Figure 4.

Confocal immunofluorescent γH2AX staining patterns in cells treated with gemcitabine and AZD1775. A) BxPC3 cells treated as described in Figure 1(a) were collected either 2 h post-radiation (7.5 Gy) or 30 h post-gemcitabine (6 h AZD1775) and sorted by γH2AX staining intensity with flow cytometry: R1, negative; R2, positive, low-intensity; or R3, positive, high-intensity. B) The percentages of cells within each gate are given for the samples shown in (A). C) Confocal immunofluorescent images of representative focal, ring and pan-nuclear γH2AX staining patterns, labeled in green. Nuclei were co-stained with propidium iodide, shown in red. D) Sorted cells were spotted on slides and scored for focal (0–10 or >10), ring, or pan-nuclear γH2AX staining. Data are from either a single control experiment (7.5 Gy condition) or are the mean ± SD of the percentage of cells with the indicated γH2AX staining pattern (n = 2 independent experiments). The numbers of cells scored for each experimental sample are given in parentheses. No cells were recovered from the 7.5 Gy, γH2AX-positive, high-intensity gate (R3).