Figure 3
Increased FANCM instability and defective chromatin recruitment of the FA core complex in PTEN-deficient cells.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 40 μg/mL cycloheximide (CHX) alone or 40 μg/mL CHX and 4 μM MG132 (CHX + MG) for the indicated times. Whole-cell lysates were prepared and immunoblotted with anti-FANCM, anti-p53, anti-PTEN, and anti-α-tubulin antibodies. RBI, Relative protein band intensity with respect to the untreated sample lane. (B) Chromatin fractionation analysis of MCF10A PTEN+/+ and PTEN−/− cells reveals a defect in the chromatin localization of FANCM and FANCA in the absence of PTEN. Cells were incubated in the absence (NT) or presence of 200 nM mitomycin C (MMC) for 24 h. W, unfractionated whole-cell lysate; S, soluble cytoplasmic and nuclear fraction; C, chromatin fraction. For (A,B), to improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. C:W, Ratio of protein in the chromatin fraction versus the whole-cell lysate. 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) Quantification of FANCM nuclear foci formation in MCF10A PTEN+/+ and PTEN−/− cells reveals a defect in MMC-inducible FANCM nuclear foci formation in PTEN−/− cells. Cells were incubated in the absence (NT) or presence of 200 nM MMC for 18 h. ***P < 0.001. (D) Quantification of FANCA nuclear foci formation in MCF10A PTEN+/+ and PTEN−/− cells reveals reduced FANCA nuclear foci formation in PTEN−/− cells both in the absence (NT) and presence of MMC. Cells were treated as described for (C). ***P < 0.001.
