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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. 4 — a Co-localization of hTR, HA-mTPP1, Flag-POT1b or Myc-POT1a on telomeres in G3 Pot1b^−/− sarcomas (white arrows). HA-mTPP1, Flag-POT1b or Myc-POT1a (FITC, green) were detected by immunostaining with anti-HA, anti-Flag or anti-Myc antibodies. hTR RNA was detected by hybridization with Cy5-hTR cDNA probes (red) and telomeres visualized by hybridization with PNA probe Cy3-OO-(CCCTAA)₄ (blue). Scale bar: 5 µm. b Quantification of co-localization of hTR with telomeres in G3 Pot1b^−/− sarcomas expressing the indicated DNAs in (a). Data show the mean ± s.d. from three independent experiments. At least 500 nuclei were analyzed per genotype. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. c Co-localization of hTR with POT1b-TPP1 or POT1a-TPP1 on telomeres in G3 Pot1b^−/− sarcoms (white arrows). Scale bar: 5 µm. d Quantification of co-localization of hTR with telomeres in G3 Pot1b^−/− sarcomas expressing the indicated DNAs in (c). Data show the mean ± s.d. from three independent experiments. At least 500 nuclei were analyzed per genotype. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. e Quantification of telomere lengths by Q-FISH in G3 Pot1b^−/− sarcomas expressing the indicated DNAs. At least 30 metaphases were analyzed per genotype. Data are from three independent experiments and box plots show the interquartile range (25 to 75%) and median from three independent experiments. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. f Telomerase processivity assay. 293T cells stably expressing mTERT and hTR were reconstituted with POT1a-TPP1, POT1b-TPP1 or the indicated mutants. The lysates were then subjected to a direct telomerase activity assay to examine telomerase processivity in the presence of POT1a-TPP1 or POT1b-TPP1. Bottom panel: Western blot indicating equal expression of POT1-TPP1 proteins used in the processivity assays.