Fig 2: Co-localization analysis.(A) LacR-ATM with SIRT6-GFP: Both are nuclear proteins that are usually found evenly spread across the entire nucleus. While we can see a sharp peak where the ATM-LacR focus is located, we do not see a peak of SIRT6-GFP at the same cellular location, which means there is no co-localization of the two proteins in this image. (B) LacR-SNF2H with SIRT6-RFP: A clear peak in the graph of the SNF2H-LacR is accompanied by a clear peak of the SIRT6-RFP, meaning positive co-localization. Since the red signal is much weaker than the green, the SIRT6 peak is much smaller than the SNF2H one. (C) LacR-Ku80 with SIRT6-RFP: In this case the SIRT6-RFP signal is much stronger that the Ku80-LacR GFP signal; however, we can still observe clear peaks at the same location. (D) LacR-GFP with SIRT6-RFP: In this negative control condition, there are no parallel peaks in the graph. However, when looking at the merged image we see that both cells are”yellow”. This just means that both signals are equally as strong (as can be seen in the graph as well) and that both proteins are spread throughout the entire nucleus; however, this is not a co-localization. (E-F) LacR-SIRT6 with 53BP1: When comparing endogenous 53BP1 (E) to the exogenous (F), we can see that using an endogenous antibody usually gives a “cleaner” higher peak with less background; however, in both cases the co-localization is clear.

Fig 3: PD-L1 expression is enhanced after irradiation of Ku80-depleted HeLa cells. (A) Immunoblotting of Ku80 in HeLa cells transfected with scrambled siCont RNA or siKu80. (B) Flow cytometry histogram of PD-L1 expression after 10 Gy irradiation of HeLa cells. Cell-surface PD-L1 expression was examined at 48 h after 10 Gy irradiation. (C) Depletion of Ku80 enhanced the up-regulation of cell-surface PD-L1 expression after irradiation. Error bars represent the standard error of three samples in the experiment. *P<0.05. si, short interfering; cont, control; PD-L1, programmed cell death-1 ligands; IR, irradiation; MFI, mean fluorescence intensity.

Fig 4: VAV2 is necessary in Ku70 and Ku80-mediated DNA NHEJ repair. a Fourteen top proteins potentially associate with VAV2 identified by mass spectrometry in ESCC cells ectopically overexpressing FLAG-tagged VAV2. Cell lysates were immunoprecipitated with antibody against FLAG and antibody against IgG was used as negative control. b Proteins potentially associate with VAV2 in KYSE30 and KYSE450 cells. Left panel is a Venn diagram showing 68 proteins identified in both cell lines. Right panel shows score of each protein. c–e Western blot detection of VAV2, Ku70 and Ku80 proteins by reciprocal immunoprecipitation with antibody against VAV2 (c), Ku70 (d) or Ku80 (e) in KYSE150 and KYSE450 cells. IgG was used as control. f Immunofluorescence analysis of VAV2 and Ku70 co-staining in KYSE150, KYSE450, PDC-4 and PDC-5 cells with or without IR (4 Gy), showing colocalization of VAV2 and Ku70. DAPI was used to label the nucleus. Scale bars, 20 µm. g Multiple immunofluorescences analysis of VAV2, Ku70, and Ku80 in clinical ESCC tumor tissues. All tumors (T) show strong colocalization signal of the 3 proteins while the corresponding non-tumor tissues (N) from patient 3, which had low VAV2, show very weak colocalization signal. Merge 1 represents the merge of VAV2 and Ku70, Merge 2 represents the merge of VAV2 and Ku80 and Merge 3 represents the merge of VAV2, Ku70, and Ku80. Scale bars, 100 µm. h Western blot analysis of FLAG-VAV2, Ku70 and Ku80 in ESCC cells ectopically expressing FLAG-tagged VAV2 and treated with siVAV2. Cell lysates were immunoprecipitated with antibody against FLAG or IgG. i Western blot analysis of VAV2, Ku70, and Ku80 in ESCC cells overexpressing VAV2. Cell lysates were immunoprecipitated with antibody against Ku70 or IgG. j Effects of increasing VAV2 expression and decreasing Ku70 expression on DNA damage in ESCC cell lines KYSE150 (upper panels) and KYSE450 (lower panels). Left panels show Ku70, Ku80, VAV2 and ?-H2AX levels detected by Western blot analysis in cells with different treatment. Middle panels are image of comet assays showing overexpression of VAV2 significantly diminished DNA damage, which could be reversed by knockdown of Ku70 expression. Right panel are the statistics of tail moments in comet assays. OE, overexpression. Data are mean ± SEM from 3 replicate experiments and 10 fields were randomly selected for each experiment. **P < 0.01; ****P < 0.0001 and ns, not significant of Student’s t-test

Fig 5: Immuno-staining of the transplanted hiPSC-RGCs in mouse retina with RGC-specific markers(A–H) Flattened whole-mount images of transplanted hiPSC-RGCs (n = 8) were co-stained with RGC-specific markers (A) BRN3, (B) RBPMS, (C) TUJ1, (D) MAP2, (E) VGLUT2, and (F) PSD95. Donor hiPSC-RGCs were also co-stained with (G) human nuclear antigen (HNA) and (H) Ku80 to confirm the donor origin of SNCG-eGFP + cells. * Since the human nuclear antigen antibody was raised in mice, some cross-reactivity with the murine retina was observed. Z-1: Zoom-1 (scale bar is 10 µm), Z-2: Zoom-2 (scale bar is 10 µm), and Z-4: Zoom-4 (scale bar is 5 µm).