Fig. 2: Identification of K879 as the PIDD1-CC SUMO-1 acceptor.

a, b ClustalW alignment of human (Hs), mouse (Mm), rat (Rn), chicken (Gg), and zebrafish (Dr) PIDD1 C termini. UPA and DD boxed in light blue and dark blue, respectively. Conserved lysine residues and ATM/ATR target T788 also indicated both in alignment (a) and AlphaFold2 model of full-length PIDD1 (b). c, d C2.Pro-BiFC HeLa cells of indicated genotypes were transfected with indicated FLAG-PIDD1 variants, treated with or without MMC and Go6976 (5 μM each) and imaged at 24 h. Arrowheads mark Venus signal, reflecting dimerization of C2 Pro-VN and C2 Pro-VC (see Supplementary Fig. 1h). At least 40 cells per sample were scored in each of n = 3 independent experiments. Representative images (c) were quantified in (d). e HeLa cells stably expressing the indicated shRNAs were transfected with indicated FLAG-PIDD1 variants, treated with or without MMC (0.1 μM) and Go6976 (1 μM) and stained with the vital dye alamarBlue at 5 days. f HeLa cells of indicated genotypes were transfected with indicated FLAG-PIDD1 variants, treated with or without MMC and Chk1 inhibitor Go6976 (1 μM each), harvested at indicated time points and analyzed by western blot. g HeLa cells of indicated genotypes were transfected with indicated FLAG-PIDD1 variants, treated with or without MMC and Go6976 (1 μM each), harvested at indicated time points, immunoprecipitated with anti-PIDD1pT788 antibodies and analyzed by western blot. h Pidd1^—/— MEF transfected with indicated FLAG-PIDD1 variants and treated with MMC and Go6976 (1 μM each) were harvested at 24 h, immunoprecipitated with anti-FLAG antibody and analyzed by western blot. Cells in (f–h) were TdR-synchronized as described in Supplementary Table 1B. Data in (d, e) expressed as means +/− SD of 3 independent experiments. **p < 0.005; ***p < 0.001; ns, non-significant; two-tailed Student’s t-test. Source data are provided as a Source data file.