(a) Cells were treated with zeocin (100 μg ml⁻¹) for 1 h. Cells were stained to detect SA-β-gal activity before (Control) and 24 h after treatment (Zeocin). Senescent BJ fibroblast cells were used as a positive staining control. (b) Zeocin treated (100 μg ml⁻¹ for 1 h) and untreated cells (Control) were subjected to cell cycle FACS analysis. Stress-induced senescent cells were used as a positive control for G2/M arrest. (c) Cells were exposed to zeocin (100 μg ml⁻¹) for 1 h and then given fresh medium for 0, 2, 4, 6 and 8 h. Cells were collected at the indicated time points and analysed by constant-field gel electrophoresis (CFGE). Telomeric fragments were detected by hybridization of their G-rich overhangs with C-rich probe under native conditions. Corresponding MW was indicated on the left. (d) Quantification of c. The relative amount of G-rich overhang on telomeric fragments was determined as the signal intensity of the smear normalized to the intensity in the entire sample. Untreated cells served as a control (Ctl). (e) Cells were exposed to zeocin (100 μg ml⁻¹) for 1 h and then given fresh medium for 0, 2, 4, 6, 8, 24 and 48 h. Untreated cells served as a control (Ctl). DDR and telomeres were visualized using antibody against 53BP1 (IF) or a probe to a telomeric sequence (FISH), respectively. Arrows indicate merged foci. Scale bar, 10 μm. (f) Quantification of e. The mean number of 53BP1 foci per cell was determined. (g) Quantification of e. The number of cells with 1 or more 53BP1 foci coincident with the signal from the telomere probe was counted, and the percentage of cells with 1 or more telomeric 53BP1 foci per cell was calculated. All values are average ±s.d. of three independent experiments (n≥100). ^**P<0.01. The Student's t-test was used to determine the statistical significance.

(a) Cells were treated with zeocin (100 μg ml⁻¹) for 48 h and then given fresh medium for 1, 2 or 3 days. DDR and telomeres were visualized using antibody against 53BP1 (IF) or a probe to a telomeric sequence (FISH), respectively. Arrows indicate merged foci. Scale bar, 10 μm. (b) Quantification of a. The mean number of 53BP1 foci per cell was determined. (c) Quantification of a. The percentage of cells with one or more telomeric 53BP1 foci per cell was determined. (d) Same as in a, except cells that senesced due to replicative exhaustion were used. Arrows indicate merged foci. Scale bar, 10 μm. (e) Quantification of d. The mean number of 53BP1 foci per cell was determined. (f) Quantification of d. The percentage of cells with one or more telomeric 53BP1 foci per cell was determined. (g) Quantification of d. Average number of telomeric 53BP1 foci in telomere dysfunction induced foci (TIF) positive cells was determined before (Ctl) and after treatment (0, 1, 2 and 3 days). All values are the average ±s.d. of three independent experiments (n≥100). *P<0.05; ^**P<0.01. The Student's t-test was used to determine the statistical significance.

(a) Schematic diagram showing the strategy used to induce DSBs in the subtelomeric region (0.5 and 1 kb from the first TTAGGG sequence in Xp/Yp). (b) Western blot analysis of Cas9 expression. Non-transfected cells served as a transfection control (Ctl) and plasmid with scrambled sgRNA (Scr) was used as a control for induction of DSBs. (c) Representative images of DSB repair foci in the subtelomere region. Scale bar, 10 μm. (d) Quantification of c. All values are the average ±s.d. of three independent experiments (n=200). ^**P<0.01. The Student's t-test was used to determine the statistical significance. (e) T7 endonuclease I (T7E1) susceptibility was used to screen and identify mutations and deletions introduced during DSBR. Cells exposed to scrambled sgRNA (Scr) were used as a control. The location of PCR primer hybridization, ∼1 kb apart, is shown. (f) A summary of the results from sequencing the PCR products.

(a) DSBs were induced at telomeres by expression of telomeric sgRNA (Telo) in CRISPR-Cas9-transfected 293T cells. Scrambled sgRNA (Scr) was used as a control. DNA was isolated and analysed by CFGE as in . (b) Immunofluorescence and FISH analysis was performed 24 h after CRISPR-Cas9 transfection of 293T cells. Normal 293T cells and cells exposed to scrambled sgRNA (Scr) were used as controls. Scale bar, 10 μm.(c) Quantification of b. The percentage of cells with one or more telomeric 53BP1 foci per cell was calculated. Values are the average ±s.d. of three independent experiments (n≥100). ^***P<0.001. The Student's t-test was used to determine the statistical significance. (d) Telomere signal at chromosome termini determined using FISH. The percentage of chromosomes with one or more telomere-free ends was calculated, based on n=1,722 or 2,922 total chromosomes for Scr or Telo-sgRNA samples, respectively. Scale bar, 20 μm. (e) Cells treated as in b were analysed for SA-β-gal activity. Stress-induced 293T senescent cells were used as a positive staining control. Approximately 1,000 cells were counted and the percentage of senescent cells was calculated.

(a) Cells were prepared as described in . Telomeres were visualized using FISH. A representative image shows large foci, indicating clustering of telomeres in cells with telomeric DSBs. Scrambled sgRNA was used as a control. Scale bar, 10 μm (b) Quantification of a, showing the average number of telomere foci in CRISPR-Cas9-targeted cells. Values are the average ±s.d. of three independent experiments (n=100). ^***P<0.001. (c) Cells were prepared as in . DNA was prepared and analysed by 2D agarose gel electrophoresis, followed by hybridization with a G-rich telomere strand-specific probe under native or denatured conditions. ExoI treatment of DNA prior to 2D gel electrophoresis eliminated the signal from ss-C-rich DNA. (d) Quantification of the C-overhang. Values are the average ±s.d. of three independent experiments. *P<0.05. (e) Quantification of the C-overhang sensitive to ExoI digestion. Values are the average ±s.d. of three independent experiments. *P<0.05. (f) Detection of T-SCE by CO-FISH (see methods). Yellow dots indicate the presence of T-SCE. Cells were transfected with telomeric sequence (Telo) or scrambled sgRNA (Scr) as indicated. Scale bar, 20 μm. (g) Summary of CO-FISH results. The Student's t-test was used to determine the statistical significance.

(a) Cells were treated and analysed as in , with the addition of B02 (13.7 μM) as indicated. (b) Quantification of a, values are the average ±s.d. of three independent experiments. The signal intensity was quantified and normalized as described for . ^**P<0.01. The Student's t-test was used to determine the statistical significance. (c) Cells were treated as in a. DNA was isolated and analysed using the in-gel hybridization assay. After electrophoresis, telomeric C-probe or G-probe was hybridized under native or denaturing conditions. (d) Schematic diagrams show hypothesized repair of DSBs in telomeres.