Figure 5.

Increased cell death and impaired cell cycle regulation of Wee1-deficient cells (A) MTT assay of Wee1^Co/-;TM-cre MEF cells that were treated with ethanol or no treatment (upper curve), or 1μM of 4-HT. (B) Apoptotic fraction of Wee1^Co/-;TM-cre MEF cells after 4-ht treatment revealed by annexin V and EtBr staining. (C) Defective G2/M cell cycle checkpoint of Wee1^Co/-;TM-cre MEF cells. Cells were treated with 1 μM 4-HT for 24 hours and fixed at 1-4 hours after they were irradiated with 10 Gy γ-ray. (D,E) Premature entry into mitosis revealed by BrdU and histone H3 double staining. Wee1^Co/-;TM-cre MEFs were incubated in the absence (D) or presence (E) of 4-HT for 24 hours prior to the staining. (F) Percentage of BrdU and histone H3 double positive cell. The percentage is calculated by the following formula [(BrdU⁺+H3Pi⁺)/H3Pi⁺]. (G) Flow cytometry analysis of mitotic cells in ethanol or 4-ht treated Wee1^Co/-;TM-cre MEFs by PI and H3Pi staining. (H) Western blotting of Wee1^Co/-;TM-cre MEFs. The arrow points to the phosphorylated form of Chk2 after lighter expossure of the gel. (I,J,K) Effect of purralanol A (10 μM) on mitotic entry of Wee1^Co/-;Tm-Cre cells in the absence (-) and presence (+) of 4-HT. Percentages of BrdU (I), H3Pi (J), and double (K) positive cells were shown.