Fig 2: The effect of TRF1 deletion on telomere volume. (A) Representative immunoblot showing deletion of TRF1 at 120 h after induction of Cre with tamoxifen in SV40LT immortalized TRF1F/F Rosa-CreERT2 MEFs. (Ctrl) Nonspecific band used as loading control. (B) Projected z-stack immunofluorescence (IF) images showing the presence of TIFs in the cells shown in A. (Green) Telomeric FISH with TelG-A647; (red) IF for 53BP1; (merge) green and red channels merged with DAPI DNA stain (blue). The percentage of telomeres with a 53BP1-positive TIF is shown below (average and SD from six experiments analyzed at 96 or 120 h). (C) Representative STORM images showing telomeric foci in cells with and without TRF1 (at 96 h after tamoxifen). Enlarged images of selected foci are shown below, and two of the enlarged images are accompanied by a localization presentation at the left that displays individual signal localizations as dots. (D–F, top) Graphs showing the natural log of Rg plotted versus the natural log of the number of telomere signal localizations obtained as in C from the indicated cells with and without Cre and processed in parallel. n = 10 cells for each condition in each independent experiment. (Bottom) Accompanying histograms of the distribution of Rg values with the means ± SDs and median values given. Cells were treated with Cre for 96 or 120 h. D–F represent three independent experiments. (G) Summary of data obtained as in D–F (and Supplemental Fig. S1) and the measured changes in average Rg values and average convex hull volumes from seven independent TRF1 deletion experiments. The mean Rg values are presented in nanometers.

Fig 3: Silencing of METTL3 enhances 5-FU-induced DNA damage in drug-resistant colorectal cancer cells. (A) Western blot assay to determine ?-H2AX levels in METTL3-KD and control HCT-8 cells following treatment with various doses (0, 40 and 60 µg/ml) of 5-FU. (B and C) Immunofluorescence staining of ?-H2AX foci and 53BP1 foci in cells. The quantification of the foci numbers per cells are presented on the right panel. (D) Comet assay of METTL3-KD and control HCT-8 cells treated with different dose of 5-FU. Representative images for the quantification of the average tail moment reported for each treatment condition. Each experiment was repeated three times. ***P<0.001. METTL3, methyltransferase-like 3; 5-FU, 5-fluorouracil; KD, knockdown; sh-, small hairpin; CN, control.

Fig 4: Knockout of PUMA elevates Rad51 in PSCs or HPCs. a Immunoblotting (IB) of Rad51, 53BP1, and PUMA expression in PUMA WT and KO PSCs or HPCs after IR. PSCs and HPCs were treated with 2 Gy IR, and then collected at 0 or 8 h post-IR for IB analysis. Actin was used as a control. b Quantification of positive cells with Rad51 foci in PUMA WT and KO PSCs at indicated time points after IR. c Representative images of Rad51 foci in PUMA WT and KO PSCs after IR. Scale bars, 10 µm. d Effects of PUMA re-expression on expression of Rad51 and 53BP1 in PUMA KO PSCs after IR. Lentivirus-mediated PUMA was infected into PUMA KO PSCs, and then selected the clones of transfected cells expressing a similar level of PUMA as that PUMA WT cells. e Effect of re-expression of PUMA on HR repair in PUMA KO PSCs. f IB of expression of Rad51 and 53BP1 in PSCs or HPCs with a Rad51 shRNA. g Effect of Rad51 knockdown on PUMA-mediated HR repair. Data are representative of three independent experiments with similar results. Error bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: Heterozygous TINF2 mutations do not cause telomere damage or genome instability.(A) Immunoblot for TIN2 and ?tubulin in control cells and the indicated clones with targeted TINF2 alleles. (B) Quantification of the immunoblot shown in A. Unpaired t-test was used to determine significance. Symbols: *p<0.05; ns, not significant (0.16). (C) Representative images of TIF analysis in control and indicated TINF2 mutant cells. IF for 53BP1 (red), telomeric FISH (green) and DNA (DAPI, blue). (D) Quantification of percentage of telomeres colocalizing with 53BP1 foci. Data from =50 nuclei per cell line, with three cell lines per genotype (with the exception of the single c.604G > C homozyg clone). (E) Representative metaphase spreads of cells with mutated TINF2 alleles. Sister telomere associations (>), telomere fusions (*), and a marker chromosome found in all clones (marker) are indicated. Telomere FISH (red), centromere FISH (green) and DNA (DAPI, gray). (F) Quantification of telomere fusions =20 spreads per cell line, with three cell lines per genotype (except for the single 604G > C homozyg clone). (G) Quantification of the % of telomeres found in sister associations. Data from =20 spreads per cell line; three cell lines per condition, except for the single 604G > C homozyg clone. For the quantification in (B), (D), (F), and (G) means ± SD and individual data points are shown. One-way ANOVA with Tukey post-test was used to determine significance, p-values: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. ns, not significant. See also Figure 4—figure supplements 1–6.