Fig 2: HUWE1 is the DNA-PKcs neddylation E3.a, b HUWE1 interacts with DNA-PK complex and UBE2M. Immunoprecipitation was performed with DNA-PKcs antibody or Flag-HUWE1, either HUWE1 or DNA-PKcs, Ku70/Ku80, UBE2M was detected by western blotting with indicated antibodies. c HUWE1 depletion abolished DNA-PKcs neddylation. HUWE1 was depleted with specific siRNAs. The depleted cells were harvested and subjected to IP with DNA-PKcs antibody following detection with NEDD8 antibody.

Fig 3: DNA-PKcs is modified by neddylation.a DNA-PKcs was neddylated after DNA damage. Either SFB- NEDD8 WT or SFB-NEDD8?G was transfected into HEK293T cells, 24 h after transfection, the cells were exposed to 10 Gy ionizing radiation. Four hours after IR treatment, cells were then harvested and subjected to Streptavidin pull-down and western blot. The blots were incubated with indicated antibodies. b MLN4924 treatment abolished the DNA-PKcs neddylation. SFB-NEDD8-transfected HEK293T cells were pretreated with 3 µm MLN4924 for 1 h, and cells were treated with 10 Gy IR and harvested for Streptavidin pull-down and western blot. c NEDP1 is required for DNA-PKcs de-neddylation. HEK293T cells were either transfected SFB-NEDD8 solely or co-transfected with NEDP1. The cells were harvested for Streptavidin pull-down and western blot 4 h after 10 Gy IR treatment. DNA-PKcs neddylation was detected by IP-western blot. d, e Endogenous DNA-PKcs was neddylated. Endogenous DNA-PKcs or endogenous NEDD8 was immunoprecipitated with specific antibodies, respectively, and the immunoprecipitated proteins were detected by either NEDD8 antibody or DNA-PKcs antibody. f Endogenous DNA-PKcs neddylation was inhibited by MLN4924 treatment. Hela cells were pretreated with 3 µm MLN4924 for 1 h and subjected for 10 Gy IR treatment. Six hours later the cells were harvested and processed to IP-western blotting.

Fig 4: DNA-PKcs neddylation promotes DNA-PKcs Ser2056 phosphorylation.a HUWE1 depletion diminished DNA-PKcs Ser2056 phosphorylation. HUWE1 shRNA-depleted cells were treated with VP-16 and harvested at indicated timepoints, then DNA-PKcs Ser2056 phosphorylation or ATM Ser1981 phosphorylation was examined in western blotting. b UBA3 knockdown abolished DNA-PKcs Ser2056 phosphorylation. UBA3 siRNA-depleted HeLa cells were treated with VP-16 and harvested at indicated timepoints, then DNA-PKcs Ser2056 phosphorylation was examined in western blotting. c, d MLN4924 treatment diminished DNA-PKcs Ser2056 phosphorylation after VP-16 or IR treatment. HeLa cells were pretreated with 3 µm MLN4924 for 1 h and subjected to VP-16 treatment or different doses of IR treatment, then the cells were harvested followed by western blotting with DNA-PKcs Ser2056 antibody or indicated antibodies.

Fig 5: DNA-PKcs neddylation modulates NHEJ repair.a Increased ?H2AX foci in HUWE1-depleted cells. shHUWE1 and control cells were treated with 10 Gy IR and stained with ?H2AX antibody at different timepoints after IR. b Depletion of HUWE1-dependent DNA-PKcs neddylation resulted in cells being arrested in G1 phase. c Depletion of HUWE1-dependent DNA-PKcs neddylation inhibited NHEJ repair. Both two HUWE1 siRNAs showed statistical significance compared with control (p < 0.05). 53BP1 knockdown was used as positive control here. d The schematic depiction of how HUWE1-dependent DNA-PKcs neddylation affects NHEJ repair.