Fig 2: M3814 over-activates IR-induced p53 apoptotic signaling in acute leukemia cells. (A) Molm-13 cells were pre-treated with M3814 (1 µM) or vehicle for 45 min before exposure to IR (2 Gy). Protein lysates were isolated 4 h after irradiation and analyzed by MSD for phospho-ATM (Ser1981) and ATM. Results shown as fold change in p-ATM normalized to total ATM protein. (B) Molm-13 cells were treated as in A. Protein lysates were isolated 4 h after irradiation and analyzed by Western blotting using antibodies for phospho-Kap1(Ser824), Kap1, phospho-CHK2(Thr68), CHK2, phospho-p53(Ser15), p53, Puma, cleaved caspase-3 and GAPDH. Panel displays cropped images from repeat blots divided and probed for different targets. Full-sized blots are presented in Supplementary Fig. 1. (C) Molm-13, Molt-4, MV4-11, THP-1 and HL-60 cells were M3814-treated and irradiated as in A. RNA was isolated 4 h post-IR and p53 target gene expression was assessed by qPCR using probes for p21, MDM2 and Puma. Expression levels were normalized to GAPDH. (D) Molm-13, Molt-4, MV4-11, THP-1 and HL-60 cells were pre-treated with increasing M3814 concentrations for 45 min before exposure to IR (1 Gy or 2 Gy). Cell growth/viability was assessed via CellTiter Glo assay on d 4. Shown are individual M3814 dose–response curves (top) and overlays of Bliss synergy on relative viability landscapes (bottom). Bliss synergy was determined using Combenefit software. (E) Molm-13, Molt-4, THP-1 and HL-60 cells were treated as in A, then exposed to 1 Gy IR and 24 h later cells undergoing apoptosis were assessed by flow cytometry using a PE-conjugated annexin V. *p < 0.05, **p < 0.01, ***p < 0.001. NS: p > 0.05.

Fig 3: DNA-Damage-Dependent ATM Phosphorylation Enhances FOXK1–53BP1 Interaction and Inhibits 53BP1 Foci Formation(A) Immunofluorescence for 53BP1 and FLAG in HeLa cells transfected with HA-FLAG-tagged WT or three mutants of FOXK1 treated with IR (10 Gy, 2 h). Representativa images of cells with strong, weak, or no 53BP1 foci are shown. Strong, ≥10 53BP1 foci; weak, <10 53BP1 foci.(B) Statistical quantification of 53BP1 foci formation from (A).(C) 293T cells co-transfected with HA-53BP1 and SFB-FOXK1 WT or mutants were immunoprecipitated with S-protein beads and subjected to immunoblot with indicated antibodies.(D) 293T cells co-transfected with HA-53BP1 and GFP-FOXK1 were irradiated (10 Gy, 2 h) and collected for immunoprecipitation with GFP-trap beads. Western blot was conducted with indicated antibodies. Quantification analysis was done with ImageJ, and the ratio of FOXK1–53BP1 interaction is shown.(E) 293T cells stably expressing FOXK1-SFB were first treated with DMSO or ATMi (AZD0156, 1 μM), ATRi (AZD6738, 1 μM), DNA-PKi (NU7441, 5 μM), or ATMi+DNA-PKi (AZD0156, 1 μM + KU7441, 5 μM) for 1 h; cells were then irradiated at 10 Gy and harvested after 2 h. Immunoprecipitation was conducted with streptavidin beads and subjected to immunoblot with indicated antibodies.The ratio of FOXK1–53BP1 interaction was normalized to the NT group and calculated as an average of three independent experiments.(F) Determination of FOXK1–53BP1 interaction in 293T WT and CHK2 KO cells in NT or IR (10 Gy, 2 h)-treated condition. The ratio was normalized to the WT NT group and calculated as an average of two independent experiments.(G) Statistical quantification of 53BP1 foci formation at different cell cycle phases in HeLa mock and overexpressing HA-FLAG FOXK1 cells.(H) 293A cells stably expressing FOXK1-SFB were synchronized and collected at different phases of the cell cycle for immunoprecipitation with streptavidin beads and subjected to immunoblot with indicated antibodies. Quantification analysis of FOXK1–53BP1 and FOXK1-pCHK2 interaction was done with ImageJ. Both ratios were calculated as an average of two independent experiments(I) Cell cycle profile of 293A cells stably expressing FOXK1-SFB at different cell cycle phases.

Fig 4: Long-term treatment with PI-103 leads to the lack of effect on Akt and DNA-PKcs phosphorylation after irradiation in K-RAS mutated cellsCells were treated with PI-103 for 1 h or 24 h and mock irradiated or irradiated with 4 Gy. Protein samples were isolated at the indicated time-points after irradiation and subjected to SDS-PAGE. The levels of P-DNA-PKcs (S2056), P-Akt (S473), P-ATM (S1981), P-Chk1 (S345) and P-Chk2 (T68) were analyzed by Western blotting. Blots were stripped and incubated with antibodies against DNA-PKcs, Akt1, ATM, Chk1 and Chk2. The densitometry values of P-DNA-PKcs/DNA-PKcs have been normalized to 1 in mock-irradiated conditions.

Fig 5: Depletion of SAMHD1 induces R-loop-mediated genome instability and cellular DNA damage response.A, Flow cytometry prolife of control U2OS and SAMHD1-depleted cells, in the absence or presence of ectopic RNaseH1-GFP expression at different time points after release from double thymidine block. Cells were subjected to PI staining followed by FACS analysis. Data represent mean ± SEM, n = 3, P values were calculated using two-way ANOVA. B, Representative images of comet assays performed under alkaline electrophoresis conditions in SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry expression. C, Quantitative analysis of comet tail moment lengths for each condition described in B. Statistical significance was assessed using two-tailed Student’s t-test (n = 50). D, Immunoblots of CHK1 and CHK2 in S phase-synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry (RNH1-mCherry) expression. Fold-induction was normalized to the empty vector-transfected control U2OS cells by quantifying the band intensity of the blots and calculating the ratios of p-CHK1 and p-CHK2 to total expression levels of CHK1 and CHK2 expression, respectively. E, Representative image of immunofluorescence assay of H2AX phosphorylated on S139 (?H2AX) in S phase synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry (RNH1-mCherry) expression. Cells were co-stained with DAPI (blue) and anti-?H2AX antibodies (green) (n = 2). F, Quantification of ?H2AX positive signal per nucleus for the immunofluorescence assay described in E. Data represent mean ± SEM, the statistical significance was assessed using the two-tailed Student’s t-test (n > 5). G, Representative images of proximity ligation assay (PLA) between PCNA and RNA polymerase II (RNAPII) in S phase synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-GFP (RNH1-GFP) expression (n = 2). Cells were subjected to PLA (red) and co-stained with DAPI (blue). H, Quantification analysis of number of PLA foci per nucleus in G. The mean value for each data is shown as a red line. Statistical significance was assessed using the two-tailed Mann-Whitney U test (n = 322).