Fig 2: hsRAD51-II3A expressing cells accumulate 53BP1 foci after treatment with HU. Representative images depicting 53BP1 foci (green) in PCNA (red) positive cells after the indicated treatments. DNA is stained in blue. Scale bar = 10 µm. Dot plot depicts quantitation of 53BP1 foci after indicated treatments. Data is combined data consisting of at least 100 nuclei from two independent experiments. Red line represents the mean. Values are presented as mean ± SD. Statistical significance was determined using the Mann–Whitney U test. Source data are provided as a source data file

Fig 3: Foci formation and disappearance. 2A. Percentage of ?H2AX-positive cell foci over time. 2B. Percentage of 53BP1-positive cell foci over time. 2C. Example of the immunofluorescence of kinetic experiment, analyzing the formation and disappearance of ?-H2AX and 53BP1 foci. Objective 40x. 2D. Evaluation of 53BP1 foci by cell lines in the Irradiation + dexamethasone condition. 2E. Evaluation of ?H2AX foci in the Irradiation + dexamethasone condition. CT2A: DEXA w/o IR (p = 0.049), DEXA IR 240' (p = 0.011); APP.PS1 L.1 w/o IR (p = 0.040), DEXA IR 240' (p = 0,000); APP.PS1 L.3 DEXA IR 240' (p = 0,044).

Fig 4: Treatment with ABT-737 increases median lifespan of progeroid mice. Starting the age of 7 months ABT-737 or DMSO-based vehicle solution was administered to LMNA+/G609G mice. a Scheme of drug administration to LMNA+/G609G mice. b Representative images depicting SA-ß-Gal activity in frozen sections of livers, pancreas, lungs, and skin from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. Scale bar, 100 µm. c Percentage of cells with SA-ß-Gal activity in liver, pancreas, lungs, and skin from ABT-737-treated and vehicle-treated LMNA+/G609G female mice. (n = 5 mice per group). d Percentages of cells stained positively for ?H2AX, p15, p53, p53BP1, and DcR2 in ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. (n = 5 mice per group). e Representative images of immunofluorescence staining for p65 (arrows) in liver sections from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. Scale bar, 50 µm. f Percentage of p65 + nuclei in livers from ABT-737-treated and vehicle-treated LMNA+/G609G female mice at the age of 11 months. (n = 5 mice per group). g Serum cytokine levels in ABT-737-treated LMNA+/G609G female mice relative to age-matched vehicle -treated LMNA+/G609G female mice. Bars represent the average intensity of 3 pooled samples (log2). Values are shown for cytokines that were decreased by more than 2-fold. h Kaplan–Meier survival curves of ABT-737-treated and vehicle-treated LMNA+/G609G mice. Data is shown for males (n = 10), females (n = 6), and both genders combined (n = 16). Chi square Gehan–Breslow–Wilcoxon Test was used for statistical analysis. i Weight curves of ABT-737-treated and vehicle-treated LMNA+/G609G mice. (n = 16 mice per group). For all graphs, values are means ± SEM. Student’s t-test was used for all comparisons between the two groups. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig 5: GLP-catalyzed H4K16me1 is associated with 53BP1 binding activity in response to DNA damage. (A) A GST pull-down assay was performed to detect the interaction between the GST-tagged Tudor domain of 53BP1 and the indicated H4 peptides. (B) A peptide pull-down assay was performed to check the interaction between the 53BP1 tandem Tudor domain and the indicated peptides. (C) HCT116 cells were treated with 40 µM etoposide for 30 min. Whole cell lysates were extracted and H4K16me1 levels were normalized to each sample before immunoprecipitation (IP) with an anti-53BP1 antibody. The interacting proteins were analyzed by western blotting. (D) Chromatin and total levels of DNA repair factors were detected in shRNA control (shCtr) or shGLP HeLa cells with or without 40 µM etoposide treatment for 30 min. A pool of shGLP#1 and shGLP#2 was used to generate the stable GLP knockdown cell line. (E) shCtr and shGLP HeLa cells were treated with 40 µM etoposide for 30 min before confocal detection of 53BP1 foci formation. 53BP1 foci numbers were counted from a minimum of 150 cells. The data represent the means ± SD. ***P < 0.001 (Student's t-test). (F) HeLa cells were pre-treated with or without 2.5 µM Bix-01294 for 24 h before X-ray irradiation. The cells were fixed at 20 min post-irradiation to detect 53BP1 foci formation by confocal microscopy. 53BP1 foci numbers were counted from a minimum of 150 cells. The data represent the means ± SD. ***P < 0.001 (Student's t-test). (G) Dynamics of GFP-53BP1 accumulation at micro-irradiated sites with or without 2.5 µM Bix-01294 treatment for 24 h. (H) Relative intensity of GFP-53BP1 at micro-irradiated sites in the experiments described in (G). The data represent the means ± SD. (I–K) shGLP cells were transfected with reconstituted Flag-GLP (WT) or Flag-GLP (?SET) for 72 h and then treated with 40 µM etoposide for 30 min. Cells were then fixed and immunostained with anti-Flag, anti-H4K16me1 and anti-53BP1 antibodies. The arrows indicate the cells with successful transfection or elevated H4K16me1 levels (I and J). Ctr, no etoposide treatment. Scale bars: 10 µm. 53BP1 foci numbers in H4K16me1 elevated or not elevated cells were counted (K). The data represent the means ± SD. ***P < 0.001 (Student's t-test).