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 ?M), 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 ?M) 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 ?M) and harvested 19 hrs post-MMC. Cells were stained with DAPI (blue, F-K), anti-?H2A.X (red, F?-K?) and anti-active caspase-3 (green, F??-K??) and imaged by confocal microscopy (0.6 ?m sections). Scale bar, 50 ?m.

(L) Quantification of ?H2A.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.