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. 2021 Sep 17;12:5514. doi: 10.1038/s41467-021-25799-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a PNA-telomere FISH shows telomeric signals present in G3 Pot1b^+/− and G3 Pot1b^−/− sarcoma cells. TelG: PNA probe Cy3-OO-(CCCTAA)₄. Red arrows point to telomere-free chromosome fusions and white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. b Distribution of telomere lengths, designated as arbitary telomere intensity, in G3 Pot1b^+/− and G3 Pot1b^−/− sarcomas determined by quantitative (Q) telomere-FISH. Telomeres from a minimum of 30 metaphases were scored in three independent experiments. Numbers indicate median telomere lengths. c Quantification of the indicated types of fusions per chromosome in Pot1b^−/− sarcomas of the indicated generations. Data show the mean ± standard deviation (s.d.) from three independent experiments. At least 30 metaphases were analyzed for each sample. d PNA-FISH of G3 Pot1b^−/− sarcoma expressing GFP, POT1a^WT, POT1b^WT or POT1b^F62A constructs. TelG: PNA probe Cy3-OO-(CCCTAA)₄. Red arrows point to telomere-free chromosome fusions; white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. e Distribution of telomere intensities by Q-FISH from metaphases in (d). A minimum of 40 metaphases were scored per cell type in three independent experiments. f Quantification of telomere-free ends in (d). Data show the mean ± s.d. from two independent experiments. At least 30 metaphases were analyzed per construct. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. g Quantification of telomere-free chromosome fusions in (d). Data show the mean ± s.d. from two independent experiments. At least 30 metaphases were analyzed per construct. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. h Detection of G-overhangs (native gel) and total telomere lengths (denature gel) by TRF Southern in G3 Pot1b^−/− sarcomas expressing the indicated constructs. Ethidium bromide (EtBr) image shows that all samples contained equal amounts of genomic DNA. Molecular weight markers are indicted. *: DNA band used for quantification. Numbers indicate relative G-overhang and total telomere signals, with telomere signals set to 1.0 for cells expressing GFP.