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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. 2 — a PNA-FISH of G3 Pot1b^−/− sarcomas reconstituted with the indicated DNA constructs. Red arrows point to telomere-free chromosome fusions; white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. b Distribution of telomere lengths by Q-FISH in (a). A minimum of 30 metaphases were analyzed in three independent experiments. Numbers indicate median telomere lengths. c Detection of G-overhangs (native gel) and total telomere lengths (denature gel) by TRF Southern. *: DNA band used for quantification. EtBr: Ethidium Bromide. Numbers indicate relative G-overhang and total telomere signals, with telomere signals set to 1.0 for cells expressing GFP. d PNA-FISH of CAG-Cre^ER; Pot1b^−/−; CTC1^F/F MEFs ± 4-HT reconstituted with indicated POT1a or POT1b. Red arrows point to telomere-free chromosome fusions and white arrows point to telomere-free chromosome ends. Scale bar: 5 µm. e Quantification of telomere lengths in (d) by Q-FISH. At least 30 metaphases were analyzed per genotype. Box show the median and interquartile range (25% to 75%) from three independent experiments. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. f Quantification of telomere-free chromosome fusions in (d). At least 30 metaphases were analyzed per genotype. Data show the mean ± s.d. from two independent experiments. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. g TRF Southern to detect the G-overhang (native gel) and total telomere lengths (denature gel) of CAG-Cre^ER; Pot1b^−/−; CTC1^F/F MEFs + 4-HT, expressing the indicated DNAs. Molecular weight markers are indicated. Numbers indicate relative G-overhang and total telomere signals, with telomere signals set to 1.0 for cells expressing GFP.