Fig 2: FOXK1 Depletion Impairs DNA Damage Response(A) Proximity ligation assay (PLA) showed the interaction of endogenous 53BP1 and FOXK1 (red spots) in non-treated (NT) and IR (10 Gy, released for 1 h)-treated cells. RIF1–53BP1 interaction was included as a positive control.(B) Quantification of PLA from (A). More than 80 cells of each group were quantified. Data are represented as mean ± SD (n = 2); ***p < 0.001, Student’s t test.(C) Generation of FOXK1 KO, FOXK2 KO, and FOXK1/2 DKO in HeLa cells. Immunoblot was performed with indicated antibodies.(D) Clonogenic survival of indicated cells after exposure to indicated doses of IR. Results are the means (± SD) of three independent experiments.(E) Cell cycle profile in WT, FOXK1 KO, FOXK2 KO, and FOXK1/2 DKO HeLa cells in NT and IR (2 Gy)-treated cells for different time points after IR.(F) Statistical quantification of the percentages of cells in different cell cycles from (E). Data are represented as mean ± SD (n = 3); **p < 0.01, ***p < 0.001, Student’s t test.

Fig 3: FOXK1 Is a Novel 53BP1-Associated Protein(A) Strategy for tandem affinity purification (TAP) in 293T 53BP1 SFB knockin cells.(B) Selected lists of 53BP1-associated proteins in soluble and chromatin fractions analyzed by mass spectrometry.(C) Interaction network of the 53BP1 soluble and chromatin-associated complexes illustrated by Cytoscape. Red, known 53BP1-binding protein; blue, putative 53BP1-binding protein.(D) Selected lists of FOXK1-associated proteins in soluble and chromatin fractions analyzed by mass spectrometry.(E) 293T cells were transfected with SFB-tagged mock control, FOXK1, or FOXK2 and then immunoprecipitated with S-protein beads and subjected to immunoblot with indicated antibodies.

Fig 4: FOXK1–53BP1 Interaction Is Required for Regulation of 53BP1-Mediated Functions(A) Western blot for expression of HA-FLAG FOXK1 WT and mutants as well as TRF2 KO efficiency in inducible TRF2 KO HeLa cells.(B) Representative images of normal telomeres and fused telomeres by metaphase DNA fluorescence in situ hybridization (FISH) with telomere probe.(C) Statistical quantification of percentage of chromosome fusions from (A). Data are represented as mean ± SD (n = 3); ***p < 0.001, Student’s t test.(D) Western blot to determine BRCA1 siRNA KD efficiency as well as the expression of FOXK1 WT and mutants in HeLa cells.(E) Statistical quantification of percentage of survival in colonies from (D) treated with olaparib at different concentrations. Data are represented as mean ± SD (n = 3); ***p < 0.001, Student’s t test.(F) Statistical quantification of percentage of cells with >10 RAD51 foci from (D). Cells were treated with 10 Gy IR and allowed to recover for 4 h. At least 200 cells were counted in each group. Data are represented as mean ± SD (n = 3); ***p < 0.001, Student’s t test.(G) Statistical quantification of percentage of survival in colonies treated with olaparib in MCF10A cells. Data are represented as mean ± SD (n = 3); **p < 0.01, Student’s t test.(H) Western blot was conducted to determine BRCA1 siRNA KD efficiency as well as expression of FOXK1 WT and mutants in MCF10A cells stably expressing GFP-tagged FOXK1 WT and mutants.(I) Determined HR repair efficiency in FOXK1 WT and mutant overexpression condition with or without BRCA1 depletion in U2OS DR-GFP reporter cells.(J) Western blot was conducted to determine BRCA1 siRNA KD efficiency as well as expression of FOXK1 WT and mutants from (I).

Fig 5: PRMT7 regulates DNA repair processes. Schematic of the DR-GFP and Ej5-GFP reporter assays. (A) DR-GFP reporter system was used to monitor HR process. Modified GFP gene (SceGFP) contains an I-SceI site in-frame. Expression of I-SceI causes DSBs at internal SceGFP sites and a non-functional GFP fragment gene (iGFP) is used to repair the DSB by HR, generating a functional GFP gene. In addition, the Ej5-GFP reporter system was used to monitor the total NHEJ process. The GFP gene is separated from its promoter by a puromycin resistance gene (puro) flanked by two I-SceI sites. Transient expression of I-SceI endonuclease causes the excision of the puro and induces the repair of the DSB by NHEJ, thus creating a functional GFP gene. (B,C) U2OS stable cell lines expressing DR-GFP or Ej5-GFP reporter constructs were co-treated (or transfected) with PRMT7 inhibitor (or siRNA) and I-SceI plasmid. After 3 days, the efficiency of HR or NHEJ was determined by counting the number of GFP-positive cells using flow cytometry. BRCA2 siRNA and Ku80 siRNA were used as positive controls to validate HR and NHEJ reporter systems, respectively. The percentage of GFP-positive cells from three independent experiments is indicated as the means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001. (D) The expression levels of HR or NHEJ-related proteins (BRCA2, Ku80, and 53BP1) in the presence of SGC8158 were determined using western blotting under the same conditions. HR: homologous recombination; DSB: double-strand break; NHEJ: non-homologous end-joining; SD: standard deviation.