Figure 2. FANCI interacts with PIDD1 and is essential for ICL-induced PIDDosome signaling.

(A) Venn diagram featuring FANCI as the sole ICL-repair protein identified in a mass spectrometry analysis of Flag-PIDD1 immunoprecipitates (Logette et al., 2011).

(B) HeLa cells treated with or without Gö6976 and MMC (1 μM each) were harvested after 24 hrs and monoclonal PIDD1 IPs were analyzed by western blot. *non-specific band.

(C) HeLa cells treated with or without Gö6976 and MMC (1 μM each) were harvested after 24hrs and monoclonal FANCI IPs were analyzed by western blot.

(D) HeLa cells grown on coverslips and synchronized as in Table S1A were treated with or without Gö6976 and MMC (1 μM each), fixed 9 hrs post-MMC, stained with the indicated antibodies and visualized by confocal microscopy (0.8 μm sections are shown). Arrowheads mark colocalization areas. Scale bar, 20 μm.

(E) Schematic diagram of FANCI (1328 a.a) highlighting the FANCD2 binding domain on its C-terminal tail (aa 1001–1328). ARM, Armadillo domain, α-α superhelix folds involved in protein-protein interactions. Leu, leucine-rich region. EDGE, EDGE motif.

(F) Schematic diagram of PIDD1-FL (910 a.a.)

(G) HeLa cells transfected with indicated Flag-FANCI constructs were harvested after 24 hrs and Flag IPs were analyzed by western blot.

(H) HeLa cells transfected with the indicated Flag-PIDD1 constructs were harvested after 24 hrs and FANCI IPs were analyzed by western blot.

(I) HCT116 cells of indicated FANCI genotypes and stably expressing shp53 were treated with Gö6976 and MMC (1 μM each) and harvested 24 hrs post-MMC. Lysates were analyzed by western blot.

(J) Patient-derived FA-I 010191 fibroblasts reconstituted with FANCI or empty vector (e.v.) were treated and analyzed as in (I).

(K) HeLa cells stably expressing the indicated shRNAs were treated with or without Gö6976 (1 μM), MMC (1 μM) or IR (10 Gy) and analyzed as in (I).

(L) HeLa cells stably expressing the indicated shRNAs were treated with Gö6976 (0.1 μM) and MMC (0.05 μM). 14 days post-treatment, cells were fixed and stained with crystal violet.

(M) Colonies from (M) of greater than 50 cells were counted. Data from 3 independent experiments expressed as means +/− SEM. *p < 0.05; **p < 0.01 ***p < 0.001, two-tailed Student’s t-test.

(N) p53^–/– zebrafish embryos of indicated fanci genotypes were treated with or without Gö6976 (1 μM) at 17 hrs post fertilization (hpf) and MMC (30 μM) 1 hr later and stained with the cell death marker acridine orange (AO) at 24 hpf. Boxed spinal-cord areas are magnified. Scale bar, 0.2 mm. As in human cells (L), zebrafish FANCI is required for death induction in embryos treated with MMC+Chk1i.

(O) Quantification of spinal cord AO stains as shown in (N). Data were collected from 2 independent experiments with at least 5 embryos scored per condition in each and reported as means +/− SEM. **p < 0.01, two-tailed Student’s t-test.