Fig 2: Que alleviated senescence and promoted self-renewal of WS hMSCs. (A) FACS analysis of hMSC-specific markers (CD73, CD90 and CD105) in vehicle- and Que-treated WS hMSCs. (B) Immunoblotting analysis of aging-related markers P16 and P21 in vehicle- and Que-treated WS hMSCs (passage 7). ß-Actin was used as the loading control. Data are shown as mean ± SEM (n = 3). *P < 0.05 vs. Ctrl; **P < 0.01 vs. Ctrl. (C) Quantitative PCR analysis of telomere length of vehicle- and Que-treated WS hMSCs (passage 7). Data are shown as mean ± SEM (n = 3). *P < 0.05 vs. Ctrl. (D) Immunostaining of Ki67 in vehicle- and Que-treated WS hMSCs (passage 7), Scale bar, 25 µm. Data are shown as mean ± SEM (cell number = 300). ***P < 0.001 vs. Ctrl. (E) Immunostaining of ?-H2AX and 53BP1 in vehicle- and Que-treated WS hMSCs (passage 7), Scale bar, 10 µm. The percentage of ?-H2AX/53BP1-double positive cells are shown as mean ± SEM (cell number = 300). *P < 0.05 vs. Ctrl. (F) FACS measurement of reactive oxygen species (ROS) by H2DCFDA in vehicle- and Que-treated WS hMSCs (passage 7). a.u., arbitrary unit. (G) RT-qPCR analysis of IL6 mRNA levels in vehicle- and Que-treated WS hMSCs (passage 7). Data are shown as mean ± SEM (n = 3). ***P < 0.001 vs. Ctrl. (H) Cell apoptosis assay in vehicle- and Que-treated WS hMSCs (passage 7). Quantitative data on the right are presented as mean ± SEM (n = 3). ***P < 0.001 vs. Ctrl

Fig 3: BLM localizes to a subset of telomeres preferentially in G2/M. (A) IF-FISH images of synchronized IMR90 cells, using a BLM specific antibody (red) and a telomeric probe, FITC-[CCCTAA]4 (green). Arrows indicate colocalization events between BLM and telomeric foci. (B) Top panel: quantification of IF-FISH data, showing the percentage of IMR90 cells with over two colocalization events. At least 100 cells were counted per time-point and values were averaged from four independent experiments. Error bars represent the standard deviation and P-values were calculated with a Student’s t-test (*P < 0.05 and **P < 0.005). Bottom panel: western blot of synchronized IMR90 cells probed with BLM antibody or a tubulin control. IMR90 lysates were collected at the indicated stages of the cell cycle. (C) Percentage of chromatids with TDs in IMR90 or BS fibroblast cell lines expressing an empty vector control, hTERT or DN_hTERT. At least 1500 chromatids per cell line, per experiment were counted from three independent experiments. (D) DAPI-positive anaphase bridges in IMR90 and BS fibroblast cells expressing an empty vector control, hTERT or DN_hTERT. At least 55 anaphases per cell line were counted from three independent experiments. (E) Percentage of IMR90 and GM02548 BS cells with over five TIF. Cells were fixed with 4% PFA and prepared for IF-FISH with a 53BP1-specific antibody and the telomeric probe FITC-[CCCTAA]4. At least 100 cells from each cell line were counted in two independent experiments. Error bars represent the standard deviation and P-values were calculated with a Student’s t-test (*P < 0.05 and **P < 0.005).

Fig 4: Interplay between e18 (RAD18-?SAP) and 53BP1 at DSBs. a Representative images showing IR-induced foci of the indicated proteins in U2OS cells transfected with vectors expressing FLAG-tagged e18, e18-D221A mutant or an empty vector control. Cells were fixed 1 h after irradiation with a 5 Gy dose and stained with antibodies that recognize the FLAG tag (green) or 53BP1 (red). Merged images with DAPI staining (blue) are also shown. Arrows indicate cells expressing e18 or e18-D221A. Scale bar: 16 µm. b Graphical representation of the percentage of FLAG-positive cells with >5 53BP1 or ?H2AX foci, under the conditions described in a. Individual experimental data are shown along with the mean ± s.e.m. (n = 3). c HDR values in HEK293T cells carrying the BFP reporter and transfected with control, 53BP1 or RNF8 siRNAs, with or without e18 expression. In all experiments, 24 h after siRNA treatment, cells were transfected with Cas9 and BFP sgRNA expression vectors, a BFP dsDNA donor, and either an empty or e18-expressing plasmid. The percentage of GFP+ cells was then determined after 72 h by flow cytometry. The values of three individual experiments were normalized to the siRNA control condition and presented along with the mean ± s.e.m. (n = 3). d HDR levels in HEK293T cells carrying the BFP reporter transfected with Cas9 and BFP sgRNA expression plasmids and a BFP dsDNA donor, in the presence of e18, i53, or both. HDR frequency was determined by quantifying the percentage of GFP+ cells, as described in c. The values of individual experiments were normalized to the control and presented along with the mean ± s.e.m. (n = 3). Statistical significance of the data shown in b–d was calculated using a one-way analysis of variance with Tukey’s multiple comparison test, with a single pooled variance (*p < 0.05, **p < 0.01, ****p < 0.0001). Source data are available in the Source Data file

Fig 5: RIF1 inhibits replication initiation after exposure to high IR doses but not after low IR doses. (A) DNA synthesis after exposure to 3 Gy IR in siCtrl and siRIF1 cells. Incorporation of EdU was measured 1 h after irradiation using a flow cytometer. (B) Schematic illustration of pulse-labeling and PCNA staining. HeLa cells synchronized in mid-S phase were pulse-labeled with EdU and stained with anti-PCNA antibodies after exposure to 0.5 Gy or 3 Gy of IR. (C,D) Time course of PCNA and EdU foci formation in non-irradiated HeLa cells. PCNA foci, scored as either colocalizing with EdU foci (>0.05 µm2 overlap area, PCNA with EdU) or not (<0.05 µm2 overlap area, PCNA w/o EdU), were quantified in each nucleus. Data are the mean±s.d. Boxes in C indicate regions shown in magnified images. Scale bar: 10 µm. (E) Transient replication block after exposure to 3 Gy of IR. After pulse-labeling, HeLa cells were irradiated with 0.5 Gy or 3 Gy and were fixed 1 or 3 h after IR exposure. Cells were stained for EdU and PCNA foci formation. PCNA foci without EdU foci were counted. (F) Restoration of the replication block after RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were irradiated with 3 Gy and were fixed 1 h after IR exposure. PCNA foci without EdU foci were counted. Data are the mean±s.d. of three independent experiments. P values indicated were calculated using a Chi-squared test. (G,H) DNA synthesis at DSB sites was measured by the colocalization of EdU with 53BP1. HeLa cells transfected with either siCtrl or siRIF1 were irradiated with 3 Gy and fixed 1 h after IR exposure. Cells were stained for EdU and 53BP1 foci formation. Frequency of 53BP1 foci colocalized with EdU were measured. Data in H are mean±s.d. and the P value indicated was calculated using a Student's t-test. Scale bar: 10 µm.