Extended Data Figure 3. The telomeric DDR is attenuated in ESCs lacking TRF2.

(a) Cartoon depicting the SILAC-PICh workflow used in (b, c). (b-c) Results of SILAC-PICh-Mass Spectrometry of Trf2^f/f ESCs (b) and Trf2^f/f EFs (c) 96 h after 4OHT or EtOH treatment. Data are shown as the fold change of heavy/light ratio for each protein in duplicate experiments for which opposite labelling with heavy/light isotopes was used. Shelterin subunits and select DNA repair factors are indicated in the plot. NA indicates protein was not detected in that experiment. (d) Venn diagram showing overlap of the proteins identified by SILAC-PICh in Wild Type Trf2^f/f ESCs and Trf2^f/f EFs (EtOH conditions). (e) IF-FISH analysis to detect γH2AX TIFs in Trf2^f/f ESCs and Trf2^f/f EFs 96 h after treatment with 4OHT or EtOH. γH2AX was detected by IF (red), telomeric DNA with FISH (white), and the DNA with DAPI (blue). Scale bar = 5 μm. (f) Quantification of the experiment shown in (e) (mean ± s.d., ≥ 300 cells/condition across 3 independent experiments, one-way ANOVA). (g) Quantification of the experiment shown in (e) (each dot represents percentage of cells with >3 TIFs in each of 3 independent experiments analysing ≥100 cells/condition per experiment, bars represent mean ± s.d. of these values, one-way ANOVA) (h) Quantification from experiment shown in Fig. 1g (each dot represents percentage of cells with >3 TIFs in each of 4 independent experiments analysing ≥100 cells/condition per experiment, bars represent mean ± s.d. of these values, one-way ANOVA). (i) Quantification of IF-FISH to detect 53BP1 TIFs in the indicated cells, 96 hours after treatment with EtOH or 4OHT. 53BP1 was detected by IF (green), telomeric DNA with FISH (red) and DNA with DAPI (blue). c1, c2 and c3 indicate 3 independently derived clones. (mean ± s.d., ≥ 200 cells/condition across 3 independent experiments, one-way ANOVA). For all panels: **** p ≤ 0.0001.