Higher DNA damage in GFI1-36N cells and lower DNA repair in GFI1-36N leukemic cells. (A) γH2AX-assay results of GFI1-36S and GFI1-36N thymocytes. γH2AX foci were stained with an antibody against γH2AX (immunofluorescence staining) and counted at different time points after 2 Gy irradiation. The images were analyzed with Imaris. Approximately 50-176 cells per sample; mean ± standard deviation (SD). P value was calculated over time. (B) Alkaline comet assay results of GFI1-36S and GFI1-36N thymocytes at different time points after irradiation with 5 Gy. Tail moment was analyzed from 34-65 cells per sample with the Comet-Assay Software from CaspLab. Mean ± SD. P value was calculated over time. (C) Murine nonleukemic progenitor cells (Lin^– cells) or (D) leukemic (MLL-AF9) BM cells from mice that received transplantation were irradiated with 3 Gy and were analyzed by flow cytometry at different time points after irradiation γH2AX level. n = 3; mean ± SD. (E) HR assay results from murine GFI1-36S- (n = 2) and GFI1-36N- (n = 2) Lin^– cells and murine GFI1-36S- (n = 2) and GFI1-36N- (n = 2) MLL-AF9 BM cells. The HR rate was measured with RT-PCR after the cells were transfected with 2 plasmids of the plasmid-based homologous recombination assay from Norgen Biotek Corp; mean ± SD (F) K562 cell lines expressing GFI1-36S or GFI1-36N were generated by CRISPR/Cas. The HR rate was measured as described in (E) 2 hours after irradiation with 3 Gy or 24 hours after treatment with 100 μg/mL temozolomide (TMZ). N = 3, mean ± SD (G) RAD51 foci formation in murine GFI1-36S and GFI1-36N leukemic (MLL-AF9) BM cells was analyzed at different time points after irradiation with 5 Gy. n = 3 (31-73 cells per sample), mean ± SD. ∗P > .05; ∗∗P < .01; ∗∗∗P < .001. (H) Cell-cycle analysis of GFI1-36S and GFI1-36N MLL-AF9 cells. GFI1-36S: n = 3 and GFI1-36N: n = 4; mean ± SD. ∗∗P < .01. No-Ir: no irradiation (0 Gy).