Figure 3. FANCI acts directly to enable ATR/ATM-mediated PIDD1 phosphorylation and PIDDosome formation.

(A) HCT116 cells of indicated FANCI genotypes and stably expressing shp53 were treated with Gö6976 and MMC (1 μM each), harvested 24 hrs post-MMC and monoclonal PIDD1 IPs were analyzed by western blot. I^–/–, FANCI^–/–.

(B) HeLa cells stably expressing the indicated shRNAs were treated with or without Gö6976 and MMC (1 μM each), lysed 24 hrs after treatment and run on a S400 HiPrep 16/60 Sephacryl column (1 ml/min). An aliquot of each fraction was concentrated and analyzed by western blot.

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

(P) (D) PC3 cells grown on coverslips and transfected with indicated siRNAs for 48hrs were treated with Gö6976 and MMC (1 μM each). Cells were fixed after 10hrs of treatment, stained using indicated antibodies and visualized by confocal microscopy (0.8 μm sections). Scale bar, 40 μm.

(E) As in (D) but substituting IR (10 Gy) for MMC. Scale bar, 40 μm.

(F) Quantification of images as in (D-E) over three independent experiments. Data shown as means +/− SEM. ***p<0.001 and ns, non-significant, two-tailed Student’s t test.

(G) HeLa cells transfected with the indicated C-terminally Flag-tagged PIDD1 constructs were lysed at 24 hpt and FANCI IPs were analyzed by western blot. WT, wild-type; T/A, T788A; T/D, T788D.

(H) HCT116 cells of indicated FANCI genotypes and stably expressing shp53 were transfected with indicated FANCI cDNAs and treated with Gö6976 and MMC (1 mM each) after 24 hrs. Cells were harvested 24hrs post-treatment and analyzed by western blot. Bottom, schematics of K523R (mono-Ub-deficient) and R1285Q (DNA binding-deficient).

(I) Model for FANCI-mediated PIDDosome signaling (see text).