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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. 3 — a Arrow points to the targeting site of CRISPR/Cas9 guide RNA of mouse mTert cDNA. TEN: Telomerase Essential N-terminal domain. TRBD: Telomerase RNA Binding Domain. RT: Reverse Transcriptase domain. CTE: C-Terminal Extension domain. b Detection of telomerase activity in G3 Pot1b^−/− parental cells and Pot1b^−/−; Tert^hypo clone #44 by the TRAP assay. 2000 cells were used in each reaction. Numbers indicate TRAP activity, with 1.0 set for G3 Pot1b^−/− samples. INC: internal control. c PNA-FISH of G3 Pot1b^−/− sarcoma and Pot1b^−/−; Tert^hypo clone #44 reconstituted with GFP or POT1b^WT. Scale bar: 5 µm. d Distribution of telomere length in (c) quantified by Q-FISH. At least 40 metaphases were analyzed per genotype in three independent experiments. Median telomere length are indicated. e Detection of G-overhang (native gel) and total telomere lengths (denature gel) in cells of the indicated genotypes by TRF Southern. *: DNA band used for quantification. EtBr: Ethidium Bromide staining shows equal loading of genomic DNA. Molecular weight markers are indicated. *: 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. f Telomere lengths in primary MEFs of the indicated genotypes measured by Q-FISH and percentage of telomere-free ends. A minimum of 40 metaphases were analyzed per genotype. At least 1500 chromosomes were scored for telomere signals and data show the mean ± s.d. from different cell lines with same genotyping. Pot1b^−/−; Tert^+/+ 3 cell lines; Pot1b^−/−; Tert^+/− 2 cell lines and Pot1b^+/−; Tert^−/− 2 cell lines. g PNA-FISH images of SV40-immortalized Pot1b^−/− or Pot1b^−/−, Tert^−/− MEFs expressing GFP or POT1b^WT. Scale bar: 5 µm. h Quantification of relative telomere lengths in (g). A minimum of 40 metaphases were analyzed per genotype. Box plots show the interquartile range (25% to 75%) and median from two independent experiments. p-values are shown and generated from unpaired Student’s t test.