Extended Data Fig. 3: No effect of CST depletion on telomere hyper-resection when 53BP1 or RIF1 are absent.
From: 53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in
a, SV40LT-immortalized Tpp1f/f53bp1−/− cells were complemented with wild-type 53BP1 or a mutant 53BP1 that lacks the ability to interact with RIF1, treated with a Stn1 shRNA as indicated and analysed by immunoblotting for 53BP1 and STN1. Representative of four experiments. b, Quantitative analysis of telomeric overhangs, as in Fig. 1c. c, Quantification of the resection at telomeres that lack TPP1, in four independent experiments performed as in Fig. 1d. d, Immunoblots showing loss of RIF1 and STN1 in the indicated Tpp1f/fRif1f/+ and Tpp1f/fRif1f/f MEFs treated with Cre (96 h) as indicated, and with or without Stn1 shRNA. Note the diminished levels of RIF1 after Cre, owing to heterozygosity in the Tpp1f/fRif1f/+ cells. e, Quantitative analysis of telomeric overhangs, as in Fig. 1c. f, Quantification of the extent of resection detected, as in e, determined from three independent experiments (indicated by different shades of grey) showing mean (centre bars) and s.d. (error bars). Each experiment involved all indicated samples analysed in parallel. g, h, Experiments to verify that the single-stranded DNA signal derives from a 3′ overhang. g, Immunoblot for STN1 and γ-tubulin in Tpp1f/fRif1f/f cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. h, Quantitative assay for telomeric overhangs, as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Welch's t-test.
