Figure 2
PTEN is required for efficient mitomycin C-inducible FANCD2 and FANCI nuclear foci formation.
(A) HCT116 PTEN+/+ and PTEN−/− cells were incubated in the absence or presence of 40 nM mitomycin C (MMC) for 18 h and FANCD2 and FANCI nuclear foci formation were analyzed by immunofluorescence microscopy. Representative images of FANCD2 and FANCI nuclear foci from cells exposed to MMC are shown. (B) Quantification of the percentage of PTEN+/+ and PTEN−/− nuclei displaying greater than five discrete FANCD2 or FANCI nuclear foci. Cells were incubated in the absence (NT) or presence of 40 nM MMC for 18 h and allowed to recover for up to 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Immunoblotting for FANCD2, FANCI, and RAD51 reveals no appreciable differences in levels of MMC-inducible FANCD2 and FANCI monoubiquitination or RAD51 between PTEN+/+ and PTEN−/− cells. Cells were incubated in the absence or presence of 200 nM MMC for 24 h and allowed to recover for up to 24 h. To improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions.
