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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. 5 — a Schematic of POT1b, POT1ab and POT1ba constructs. All 5 amino acids in POT1b required to interact with CTC1 are shown. b Quantification of co-localization of hTR with telomeres in G3 Pot1b^−/− sarcomas expressing the indicated DNAs. Data show the mean ± s.d. from three independent experiments. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. c Quantification of telomere lengths in G3 Pot1b^−/− sarcomas expressing the indicated DNAs. At least 40 metaphases were analyzed per genotype. Box 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. d TRF Southern blot to detect G-overhangs (native) and total telomeres (denature) in G3 Pot1b^−/− sarcomas expressing the indicated DNAs. *: DNA band used for quantification. EtBr: Ethidium Bromide staining reveals equal loading of genomic DNA. Numbers indicate relative G-overhang and total telomere signals, with telomere signals set to 1.0 for cells expressing GFP. Molecular weights are indicated. e WT MEFs expressing cell-cycle sensors Geminin (blue) or CDT1 (blue) were reconstituted with the indicated POT1 proteins (green). Endogenous TRF2 marked telomeres (red). White arrows indicate POT1/TRF2 co-localization. Scale bar: 5 µm. f Localization of POT1a, POT1a-TPP1, POT1b and POT1b-TPP1 on telomeres during S/G2 (Geminin positive, left panel) and during G1 (CDT1 positive, right panel). Data show the mean ± s.d. from three independent experiments. At least 200 nuclei were analyzed for each genotype. g Endogenous STN1 localizated to telomere in wild-type MEFs expressing POT1a-TPP1 or POT1b-TPP1. STN1 was immunostained with anti-STN1 antibody (green) and telomeres were detected by PNA FISH with Cy3-OO-(CCCTAA)₄ (red). Nuclei were stained with DAPI (blue). Scale bar: 5 µm. h Quantification of (g). Data show the mean ± s.d. from three independent experiments. 200 nuclei were analyzed in two independent experiments. p-values are shown and generated from one-way ANOVA analysis followed by Tukey’s multiple comparison. i POT1a-TPP1 expression represses telomerase recruitment to telomeres. Quantification of telomerase localization to telomere in WT MEFs expressing (+) POT1a-TPP1 or vector control (−). At least 200 nuclei were analyzed for each group and data show the mean ± s.d. from two independent experiments. p-values are shown and calculated from unpaired Student’s t test.