Extended Data Fig. 5: Localization of TRF1FL and TRF1ΔE8.
From: Nonsense-mediated mRNA decay safeguards telomeres in pluripotent stem cells
a, Immunofluorescence combined with FISH (IF-FISH) showing the expression and localization of Myc-tagged inducible TRF1 constructs (red) and telomeric DNA (TTAGGG, green) in cells with stable integration of either full-length TRF1 (iTRF1FL) or TRF1 lacking exon 8 (iTRF1ΔE8). Cells were treated with doxycycline (DOX) to induce construct expression or with DMSO as a control. More than 155 cells per genotype were scored. Details on the exact cell number per genotype are in Source Data File. b, IF-FISH for Myc (red) and telomeric DNA (TTAGGG, green) performed in Smg6−/− or Upf1HM ESCs stably expressing full-length inducible TRF1 (iTRF1FL), treated with either DMSO or doxycycline (DOX). More than 201 cells per genotype were scored. Details on the exact cell number per genotype are in Source Data File. c, Metaphase spreads from Upf1HM and Upf1HM Trf2−/− ESCs either untreated (DMSO) or expressing ectopic full-length TRF1 (TRF1FL) (DOX). Quantification of telomeric fusions is shown. More than 248 chromosomes per genotype (n = 1 biological replicate) were scored. Details on the exact cell number per genotype are in Source Data File. d, Metaphase spreads from Trf2f/f and Trf2f/f Trf2dTAG ESCs either untreated (DMSO) or treated with OHT and dTAG. Quantification of telomeric fusions is shown (e). More than 834 chromosomes per genotype (n = 3 biological replicates) were scored. Details on the exact cell number per genotype are in Source Data File. Statistical analysis by one-way ANOVA; ns, not significant, P = 0.6850, ****P = 0.00001. f, Representative Immunofluorescence-Fluorescence In Situ Hybridization (IF–FISH) showing gH2AX (red) and telomeric DNA (green) in Trf2f/f (Ctrl.) and Trf2f/f Trf2dTAG ESCs with or without 4-hydroxytamoxifen (OHT) treatment to induce TRF2 depletion, and without or with dTAG treatment ( + dTAG) to induce TRF1 depletion. g, Quantification of telomere dysfunction-induced foci (TIFs) in (f), defined as ≥10 gH2AX foci colocalizing with telomeres. Data are mean ± s.d. from three (n = 3) biological replicates. Statistical analysis by one-way ANOVA; *P = 0.0342; ***P = 0.001. More than 155 cells per genotype were scored. Details on the exact cell number per genotype are in Source Data File. h, Immunofluorescence for Myc (red) in control Trf1dTAG ESCs (Ctrl.), or Trf1dTAG cells stably expressing full-length inducible TRF1 (iTRF1FL) or truncated TRF1 (iTRF1DE8), treated with either DMSO or doxycycline (DOX). i, IF-FISH for FLAG (red) and telomeric DNA (TTAGGG, green) performed in cells of indicated genotypes and under conditions as in (h).
