Fig. 4

Combined inhibition of Wee1 and Chk1/2 leads to increased mitotic entry and DNA-damage in S-phase cells. a. Cells were treated with MK1775 (200nM) and/or AZD7762 (100nM) for 48 h. Expression of proteins involved in DNA-damage response and apoptosis was determined by western blot analysis, and α-tubulin was used as loading control. b, c, d and e. WM983B cells were treated with either DMSO (control) or high concentrations of MK1775 (2 μM) and/or AZD7762 (1 μM) for 3 h, and analyzed by Flow Cytometry B. Subpopulation gating of mitotic (blue) and pre-mitotic (purple) cells were based on their DNA content and expression of pHH3. The percentages of cells are indicated above the gated populations. c. The percentage of cells in mitosis (M) and pre-mitosis (Pre-M) are shown as fold change from the control cells. d. Expression of γ-H2A.X, indicative of DNA-damage, was assessed following treatment with inhibitors. e. The median expression of γ-H2A.X was determined for cells in the indicated phases of the cell cycle (for gating see Additional file 3: Figure S3), and shown as fold change from the control cells. Data are representative or average of four independent experiments