Fig. 3.

hCINAP regulates NPM1 deSUMOylation in a SENP3-dependent manner during DSB repair. a, b The effect of damage on NPM1 deSUMOylation was investigated using a His-SUMO pull-down assay. Nickel precipitation (Ni²⁺) from His-SUMO3-expressing U2OS cells treated with IR at different time points is shown. The level of NPM1 SUMOylation was assessed using an anti-NPM1 antibody. c, d hCINAP^WT and hCINAP^−/− HEK293T cells were transfected with His-SUMO3 for 48 h and then treated without or with IR (10 Gy). Cells were collected at different time points after IR treatment (c, 8 h; d, a time course is shown in the Fig.) and subjected to His-SUMO pull-down analysis to access the level of NPM1 SUMOylation. e The densitometry analysis was done on three independent His pull-down experiments of Fig. 3d. f, g Dynamic accumulation of NPM1 and hCINAP at DNA-damage sites in living cells. U2OS cells expressing GFP-hCINAP and mCherry-hCINAP were monitored after micro-irradiation via time-lapse fluorescence microscopy f. Quantifications of GFP-hCINAP and mCherry-hCINAP accumulation at laser track sites were performed using ImageJ software g. The fluorescence intensity values in the micro-irradiated areas were pooled from 10 independent cells and plotted vs. time. h HEK293T cells were transfected with Myc-SENP3 and Flag-hCINAP for 48 h. Co-IP assays were then performed using an anti-Myc antibody. The bound complexes were analyzed via immunoblotting. i HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to co-IP analyses to examine the effects of hCINAP on the interaction between SENP3 and NPM1 using the indicated antibodies. j hCINAP^−/− HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to co-IP analyses to examine the effects of hCINAP on the interaction between SENP3 and NPM1 using the indicated antibodies. k The SENP3^−/− cell line was constructed using the CRISPR/Cas9 system as shown in Supplementary Fig. 2c. Knockout efficiency was determined by immunoblotting using an anti-SENP3 antibody (Supplementary Fig. 2d). The level of endogenous NPM1 SUMOylation was monitored via His-SUMO pull-down analysis in SENP3^WT and SENP3^−/− cells. The densitometry analysis was carried out on three independent His pull-down experiments. Unprocessed scans of blots are provided in Supplementary Fig. 13