Fig. 7

(A) MDA-MB-231 and SAS cells treated with indicated doses of MMC and harvested at 9 hrs post-treatment. FANCI IPs were analyzed by western blot. Ub, monoubiquitinated form.

(B-D) HeLa cells synchronized as in Table S1B were transfected with indicated siRNAs and treated with MMC (1 μM), harvested at 7.5 hrs post-treatment, immunoprecipitated with monoclonal PIDD1 (B-C) and FANCI (D) antibodies and analyzed by western blot.

(E) FANCI^–/– HCT116 cells transfected with sip53 were synchronized and reconstituted with indicated Flag-FANCI cDNAs as in Table S1C, treated with MMC (1 μM) and harvested 9 hrs later. FANCI IPs were analyzed by western blot. Cleaved C3 (cl.C3) and PARP (cl.) were assessed in 24 hr lysates.

(F-K’’’) FANCI^–/– HCT116 cells stably expressing shp53 were grown on cover slips, synchronized, reconstituted with indicated Flag-FANCI cDNAs, treated with or without MMC (1 μM) and harvested 19 hrs post-MMC. Cells were stained with DAPI (blue, F-K), anti-γH2A.X (red, F’-K’) and anti-active caspase-3 (green, F’’-K’’) and imaged by confocal microscopy (0.6 μm sections). Scale bar, 50 μm.

(L) Quantification of γH2A.X and/or active caspase-3 positive cells, as shown in (F-K’’’), over three independent experiments. Data expressed as means +/−SD, ***p < 0.001, two-tailed Student’s t-test.

(M) Model for the regulation of FANCI switch function by K523 monoubiquitination.