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. 2023 Apr 14;14:2144. doi: 10.1038/s41467-023-37761-w

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© The Author(s) 2023, corrected publication 2023

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. 1 — a PNA- FISH of interphase nuclei showing time-dependent increase of CCCTAA-positive telomere filaments (green arrowheads) and UT foci (white arrowheads) in Rap1^+/+ and Rap1^–/– MEFs expressing the indicated DNAs. CCCTAA-positive filaments and telomeres detected with TelC-Cy3 (CCCTAA)₃ PNA telomere probe (red) and DAPI to stain nuclei (blue). Inset: magnified view of CCCTAA-positive filaments and UTs. Scale bars: 5 µm. b Quantification of UTs in Rap1^+/+ and Rap1^–/– MEFs after 120 hrs. The mean of three independent experiments ± SD are shown, at least 200 nuclei analyzed per experiment. *P = 0.0458, ****P < 0.0001 by one-way ANOVA. ns: non-significant. c Quantification of UTs in Rap1^–/– MEFs expressing TRF2^ΔB at the indicated time points. The mean of three independent experiments ± SD are shown, at least 250 nuclei analyzed per experiment. *P = 0.0240, ***P = 0.0010, ****P < 0.0001 by one-way ANOVA. ns non-significant. d Representative images of UTs from two independent experiments shown to co-localized (white arrowheads) with indicated chromosomal specific sub-telomere probes (green) in U2OS cells expressing TRF2^∆B;L288R. Scale bars: 5 µm. e Quantification of CCCTAA-positive filaments in Rap1^–/– MEFs expressing TRF2^ΔB at the indicated time points. The mean of three independent experiments ± SD are shown; at least 250 nuclei analyzed per experiment. **P = 0.0060, ***P = 0.0003, ****P < 0.0001 by one-way ANOVA. ns non-significant. f Representative PNA-FISH on metaphase spreads of Rap1^–/– MEFs expressing TRF2^ΔB from three independent experiments showing maximum formation of CCCTAA-positive telomere filaments (red) at 48 hrs (green arrowheads), UTs (white arrowheads), signal free ends (*), chromosomes fused with telomeres (orange arrowheads), chromosomes fused without telomeres (pink arrowheads). A minimum of 35 metaphase for each sample were examined per experiment. Scale bars: 15 µm. g The Fucci assay showing UTs formed primarily during the S-phase in U2OS cells. The mean of three independent experiments ±SD are shown, at least 150 nuclei analyzed per experiment. **P = 0.0015 by two-tailed unpaired t test. h Quantification of p-RPA32 and RAD51 localization on telomere bridges in Rap1^–/– MEFs expressing indicated DNA constructs after 48 h. The mean of two independent experiments ± SD are shown, at least 200 nuclei analyzed per experiment. ****P < 0.0001 by one-way ANOVA. ns non-significant. i Quantification of UT frequencies in 53Bp1^+/+ and 53Bp1^–/– MEFs reconstituted with WT 53BP1 and the indicated 53BP1 mutants in the presence of shTrf2 + TRF2^ΔB;L286R. The mean of two independent experiments ± SD are shown, at least 150 nuclei analyzed per experiment. ***P = 0.0010, ****P < 0.0001 by one-way ANOVA. Source data are provided as a Source Data file.