Fig. 6

PKMYT1-induced NPM1 S260 phosphorylation regulates NPM1 deSUMOylation during DSB repair. a U2OS cells were transfected with indicated siRNAs for 48 h before harvesting the cells for Western blot. b, c U2OS cells were transfected with indicated plasmids and subjected to His-SUMO pull-down analysis to access the level of NPM1 SUMOylation. d U2OS cells were transfected with indicated plasmids and siRNAs and subjected to His-SUMO pull-down analysis. e U2OS cells were transfected with indicated plasmids for 24 h and treated with or without RP6306 (2 μM) for 24 h. Cells were collected and subjected to His-SUMO pull-down analysis to assess the level of NPM1 SUMOylation. f 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 and subjected to His-SUMO pull-down analysis to access the level of NPM1 SUMOylation. g HEK293T cells were transfected with His-SUMO3 for 48 h and then treated without or with cisplatin (2 μM). Cells were collected at different time points after cisplatin treatment and subjected to His-SUMO pull-down analysis. h HEK293T cells were transfected with His-SUMO3 and either siNC or siPKMYT1 for 48 h and then treated without or with cisplatin (2 μM). Cells were collected at different time points after cisplatin treatment and subjected to His-SUMO3 pull-down analysis. i HEK293T cells were transfected with His-SUMO3 for 48 h and then treated without or with RP6306 (2 μM) for 24 h. Cells were collected at different time points after cisplatin (1 μM) treatment and subjected to His-SUMO3 pull-down analysis. j HEK293T cells were co-transfected with His-SUMO3 for 48 h and either NPM1 WT or NPM1 S260A for 24 h, followed by DDP treatment. Cells were collected at various time points to investigate the influence of NPM1 S260 phosphorylation on NPM1 SUMOylation