Figure 1. ICLs trigger PIDDosome signaling.
(A) HeLa cells stably expressing the indicated shRNAs, treated with or without Gö6976 and MMC (1 μM each) and harvested 24hrs post-MMC were analyzed by western blot. pro-C2, procaspase-2; p35, intermediate cleavage fragment; p19, mature product. FL, full-length; C, large C-terminal autocleavage product; CC, short, distal, C-terminal autocleavage product (see J).
(B) HeLa cells of indicated genotypes stably expressing C2 Pro-BiFC and mCherry were treated with Gö6976 and MMC (5μM each) plus VD-OPH (20 μM) and fixed at 24 hours. Confocal images (5 μm sections) show C2 BiFC (yellow) and mCherry (red) expression. Scale bar, 20 μm.
(C) Quantification of images as in (B) over 3 independent experiments, with data expressed as means +/− SD. **p < 0.01, two-tailed Student’s t-test.
(D) HeLa cells stably expressing the indicated shRNAs were treated with or without Gö6976 and MMC (1 μM each). Cells were stained with the vital dye alamarBlue 72 hr post-treatment. Data are means +/− SD of 3 independent experiments. **p < 0.01, two-tailed Student’s t-test.
(E) HeLa cells initially treated with ATM inhibitor (KU55933) and ATR inhibitor (ETP46464), 10 μM each, were treated with or without Gö6976 and MMC (1 μM each) or IR (10 Gy) after 1 hr. Cells were harvested 24hrs post-treatment and analyzed by western blot.
(F-I) HCT116 cells of indicated FANCI genotypes and stably expressing shp53 were treated with Gö6976 and MMC (1 μM each) and harvested at 0 hrs, 9 hrs and 24 hrs post-MMC. Lysates (I) were immunoprecipitated with s-17 antibody to the PIDD1 N-terminus (F) or monoclonal anti-ATR (G) and anti-ATM (H) antibodies and analyzed by western blot.
(J) Schematic diagram of full-length (FL) PIDD1 (910 a.a.) and autoproteolytic cleavage products PIDD1-N, PIDD1-C and PIDD1-CC. LRR, leucine-rich repeats; ZU-5, ZO-1 and Unc5-like domain; UPA, uncharacterized protein domain in UNC5, PIDD and Ankyrin, implicated in PIDD1 oligomerization (Janssens and Tinel, 2012); DD, death domain.
(K) HeLa cells transfected with the indicated Flag-tagged PIDD deletion constructs were harvested 24hrs post-transfection. Flag IPs were analyzed by western blot.
(L) Recombinant GST-PIDD1-CC proteins (WT and T788A phosphomutant, T/A) were incubated with Flag-ATR and His-ATRIP for an in vitro kinase assay. Reactions were analyzed by Coomassie staining and western blot. α-pSQ/TQ, polyclonal antibody to phosphorylated SQ or TQ motifs, such as T788Q789 in PIDD1 (Ando et al., 2012). *, non-specific band.
(M) Schematic of PIDD1 phosphorylation by ATR and ATM in response to ICLs and DSBs. ATM plays a delayed role in the ICL response (dotted).