Fig. 3: PIDD1 SUMOylation is catalyzed by PIAS1 and reversed by SENP3. | Nature Communications

Fig. 3: PIDD1 SUMOylation is catalyzed by PIAS1 and reversed by SENP3.

From: Stepwise phosphorylation and SUMOylation of PIDD1 drive PIDDosome assembly in response to DNA repair failure

Fig. 3

a–c C2.Pro-BiFC HeLa cells transfected with indicated siRNAs were treated with or without MMC and Go6976 (5 μM each) and analyzed by confocal microscopy at 24 h. At least 40 cells per sample were scored in each of n = 3 independent experiments. Representative images (a) were quantified for percentage of C2 BiFC positive cells (b) and overall signal intensity (c). d HeLa cells treated with MMC and Go6976 and harvested at indicated time points were immunoprecipitated with anti-PIDD1pT788 antibody and analyzed by western blot. e HeLa cells transfected with indicated siRNAs were treated with MMC and Go6976, harvested at indicated time points and analyzed by western blot. f HeLa cells transfected with indicated siRNAs, co-transfected with indicated Myc-PIAS1 constructs and treated and analyzed as in (e). C1, catalytically inactive. (g) HeLa cells transfected with indicated Myc-PIAS constructs and treated with MMC and Go6976 were analyzed as in (e). CCpT788di-SUMO1, di-SUMO-1ylated CCpT788. h FANCP—/— HeLa cells treated with MMC were harvested at indicated time points and PIDD1pT788 immunoprecipitates were analyzed by western blot. i–k Cells treated and analyzed as in (a) were quantified for percentage of C2 BiFC positive cells (j) and average number of Venus puncta per cell (k). l Parent and SENP3—/— HeLa cells treated with MMC and Go6976 at indicated doses were analyzed as in (i–k). At least 40 cells per sample were scored in each of n = 2 independent experiments (representative images shown in Supplementary Fig. 3c) (m) Cells as in (l) were stained with the vital dye alamarBlue at 5 days. n HeLa cells transfected with indicated siRNAs and treated with indicated drugs were immunoprecipitated with anti-PIDD1pT788 antibody and analyzed by western blot. Cells in (d–h, n) were TdR-synchronized as described in Supplementary Tables 1A (d, h), 1B (e–g), 1C (n). Unless otherwise indicated, drugs were given at 1μM. Data in (b, c, j, k, m) are expressed as means +/− SD, with *p < 0.05, **p < 0.005, ***p < 0.001, ns, non-significant, two-tailed Student’s t-test. Source data are provided as a Source data file.

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