Fig 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.

Fig 3: PTEN function in ICL repair is protein phosphatase and SUMOylation-dependent.(A) HCT116 PTEN-/- cells were stably transduced with pLenti6.2-LacZ, -PTEN-WT, -PTEN-C124S, -PTEN-G129E, and -PTEN-K254R. Whole-cell lysates were prepared and immunoblotted for PTEN, AKT, AKT pS473, RAD51, and a-tubulin. To improve clarity and conciseness, the presented blots have been cropped. All gels were run under the same experimental conditions. (B) PTEN-C124S and PTEN-K254R fail to rescue the sensitivity of PTEN-/- cells to the clastogenic effects of mitomycin C (MMC), in contrast to PTEN-WT or PTEN-G129E. Cells were incubated in the absence (NT) or presence of 20 nM MMC for 24 h and metaphase spreads were analyzed for structural chromosome aberrations. ***P < 0.001. (C,D) Defective FANCD2 (C) and FANCI (D) nuclear foci formation in PTEN-/- cells expressing LacZ, PTEN-C124S, and PTEN-K254R. Cells were incubated in the absence (NT) or presence of 200 nM MMC for 18 h. ***P < 0.001.

Fig 4: The HMTi BRD4770 induces the monoubiquitination of FANCD2 and FANCI(A) and (B), U2OS (A) and BJ-TERT (B) cells were incubated in the absence (NT) or presence of 10 µM BRD4770, 2.5 µM BIX01294, 5 µM GSK-J1 and 1 µM PBIT, with (+) and without (-) 200 nM mitomycin C (MMC) for 24 h. Whole-cell lysates were prepared and immunoblotted with anti-FANCD2, anti-FANCI, and anti-a-Tubulin antibodies. (C) U2OS cells were incubated with the indicated concentrations of BRD4770 and BIX01294 for 24 h, and whole-cell lysates were immunoblotted with anti-FANCD2, anti-FANCI, and anti-a-Tubulin antibodies. L:S Ratio, ratio of monoubiquitinated to nonubiquitinated FANCD2 or FANCI.

Fig 5: Inhibition of class I and II HDACs attenuates FANCD2 and FANCI monoubiquitination in BJ-TERT cells(A) and (B), HeLa cells were pre-treated with the indicated concentrations of trichostatin A (TSA) (A) or vorinostat (SAHA) (B) for 4 h, followed by co-incubation with (+) and without (-) 200 nM MMC for a further 20 h. Whole-cell lysates were prepared and immunoblotted with anti-FANCD2, anti-FANCI, anti-CHK1 pS345, anti-CHK2 pT68, anti-H4K16ac, and anti-a-Tubulin antibodies. (C) and (D), BJ-TERT cells were treated identically to that described for HeLa cells above. L:S Ratio, ratio of monoubiquitinated to nonubiquitinated FANCD2 or FANCI.