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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. 6 — a Interaction interfaces between POT1 and TPP1. POT1: green; POT1a: cyan; POT1b: magenta; hTPP1/mTPP1: black. Top three panels show the POT1/POT1a/POT1b interactions around residues Q286^hTPP1/R180^mTPP1. The bottom left panel shows hydrophobic interactions around residues W293^hTPP1/W205^mTPP1, and the bottom right panel shows hydrophobic interactions around residues Y306^hTPP1/F218^mTPP1. b Table of amino acids from (a) predicted to form direct interactions between POT1 and TPP1. c Schematic of point mutations generated to change specific residues in POT1a or POT1b. d Co-IP assay reveal that most POT1b mutants interacts poorly with TPP1. e Quantification of co-localization of hTR with telomeres in G3 Pot1b^−/− sarcomas reconstituted with the indicated POT1a and POT1b DNAs. 500 nuclei were analyzed per genotype. Data show the mean ± s.d. from three independent experiments. f Quantification of telomere lengths by Q-FISH in G3 Pot1b^−/− sarcomas expressing POT1a or POT1b constructs. 30 metaphases were analyzed per construct. Box show the median and interquartile range (25% to 75%) from three independent experiments. g Co-IP assay revealed that POT1a mutants interact robustly with TPP1. h Quantification of co-localization of hTR with telomeres in G3 Pot1b^−/− sarcomas reconstituted with the indicated POT1a and POT1b constructs. 500 nuclei were analyzed per genotype. Data show the mean ± s.d. from three independent experiments. i Quantification of telomere lengths by Q-FISH in G3 Pot1b^−/− sarcomas expressing POT1a or POT1b DNAs. 30 metaphases were analyzed per construct. Box show the median and interquartile range (25% to 75%) from three independent experiments. j Quantification of co-localization of hTR with telomeres in MMTV-Cre; p53^Δ/Δ; Pot1a^Δ/Δ cells reconstituted with POT1a constructs. 500 nuclei were analyzed per genotype. Data show the mean ± s.d. from three independent experiments. k Q-FISH quantification of telomere length in MMTV-Cre; p53^Δ/Δ; Pot1a^Δ/Δ tumor cells reconstituted with the indicated DNAs. 40 metaphases were analyzed. Box show the median and interquartile range (25–75%) from three independent experiments. For panels (e, f, h–k), p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison.