Figure 2

Elevated expression of CDC25B in S-phase induces γ-H2AX labeling. (A) Asynchronous U2OS cells overexpressing Ha-CDC25B (U2OS CDC25B) (or not) were subjected to immunofluorescence analysis after staining with anti-Ha and anti-γ-H2AX antibodies (bar = 10 μM). In the upper right part of A, a western blot analysis of Ha-CDC25B and endogenous CDC25B levels using anti-CDC25B antibodies. In the lower panel, after synchronization by nocodazole treatment and mitotic shake off followed (-tet) or not (+tet) by CDC25B induction as described in figure 1B, the cells were processed for western blot using antibodies against γ-H2AX and Ha tag (bar = 10 μM). (B) Asynchronous U2OS cells expressing Ha-CDC25B (U2OS CDC25B) or not (U2OS) were subjected to BrdU labeling for 15 min (30 μM), then processed for immunofluorescence analysis using antibodies against γ-H2AX and BrdU. (bar = 10 μM). (C) Asynchronous U2OS cells expressing Ha-CDC25B were processed for flow cytometry analysis with γ-H2AX antibodies and propidium iodide. The % indicates the quantification of γ-H2AX labeling in the global population (left panel) and in each phase of the cell cycle (right panel). Color code in flow cytometry: blue>red>green. (D) HCT116 and HCT116 CDC25B+ with a slightly elevated level of Ha-CDC25B were blocked by thymidine (2.5 mM) for 17 h then released in DMEM for 3 h. The cells were processed for immunofluorescence using antibodies against γ-H2AX and Ha-tag and for western blotting using antibodies against CDC25B, Ha-tag, γ-H2AX and actin as loading marker (bar = 10 μM). Frequency histogram from 2 immunofluorescence analyses, shows the distribution of γ-H2AX fluorescence intensity in the cells (t-test, parental HCT116 (black bars) compared to HCT116 CDC25B+ (red bars).