Figure 4
Inhibition of PLK1 rescues defective FANCD2 nuclear foci formation but is not sufficient to rescue the chromosome instability of PTEN−/− cells.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence (−) and presence (+) of 100 nM BI2536 and 500 nM mitomycin C (MMC) for 18 h, and whole-cell lysates were immunoblotted with the indicated antibodies. L:S, Ratio of phosphorylated to unphosphorylated FANCM. (B) PTEN+/+ and PTEN−/− cells were incubated in the absence and presence of 200 nM MMC for 24 h. Whole-cell lysates were then incubated in the absence (−) or presence (+) of 10 U/μg λ-phosphatase for 4 h at 30 °C, followed by immunoblotting with the indicated antibodies. For (A,B), to improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. RBI, Relative unmodified FANCM protein band intensity. Immunoblotting experiments were performed multiple times with similar results. Protein band quantifications are from the immunoblots shown and are representative of results from several experiments. (C) PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 2 nM BI2536, 20 nM MMC, or both BI2536 and MMC for 24 h and metaphase spreads were analyzed for the presence of numerical and structural chromosome aberrations. (D) PTEN−/− cells stably expressing empty vector or wild-type PTEN were incubated in the absence or presence of 5 nM BI2536, 200 nM MMC, or both BI2536 and MMC for 24 h, and allowed to recover for 8 h, and FANCD2 nuclear foci formation were analyzed by immunofluorescence microscopy. *P < 0.05; ***P < 0.001.
