Figure 3

WEE1 inhibition abrogates the γ-irradiation induced G ₂ /M cell cycle arrest in OS cells and leads to mitotic catastrophe. (A) Histograms of FACS cell cycle analysis of OS cells treated with 4 Gy IR, 0.5 μM WEE1-inhibitor, and combination treatment. Percentages of cells in G₂/M phase and the mitotic index (MI) in percentages are shown. After irradiation, the percentages of cells in G₂/M phase increased, whereas the percentages of mitotic cells remained unaltered. After subsequent treatment with WEE1-inhibitor, the G₂ arrest is completely abrogated; the percentages of cells in G₂/M phase return to control values, while the percentages of mitotic cells increase dramatically. This indicates a forced progression through the G₂ cell cycle checkpoint into mitosis. (B) Fluorescence microscopy images of nuclei (blue) with ionizing radiation induced foci (IRIF) (green) indicating DNA damage, visualized using immunofluorescent γ-histone-H2AX staining of DNA breaks at 1 h and 24 h post-irradiation. Cells treated with IR alone show fewer IRIF at 24 h after treatment than cells treated with IR + WEE1-inhibitor. (C) Caspase activation in OS cells treated with 4 Gy IR or 4 Gy IR + 0.5 μM WEE1-inhibitor 6 h and 24 h after treatment. Bars represent experiments performed in triplicate; error bars indicate SD. After 6 h, there is a mild induction of caspase activity. Caspase activation levels are comparable between the two treatment groups. At the 24 h time point there is a significantly higher caspase activation in the cells treated with combination treatment for all cell lines (student's t-test: p < 0.01).