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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. 4 — a PNA-FISH on metaphase spreads showing CCCTAA-positive filaments (green arrowheads) and UTs (white arrowheads) in Rap1^–/– MEFs expressing TRF2^ΔB with + /– 0.5 µM aphidicolin (APH) treatment. Scale bars: 20 µm. A minimum of 30 metaphases for each sample were examined per experiment. b Quantification of UT frequencies in the interphase nuclei of Rap1^+/+ and Rap1^–/– MEFs expressing the indicated DNA constructs in (a). Mean of three independent experiments ± SD are shown from a minimum 250 nuclei analyzed per experiment. ****P < 0.0001 by one-way ANOVA. ns non-significant. c Representative IF-FISH images showing UTs co-localized to SMARCAL1 (green) in U2OS cells expressing shTRF2 + TRF2^ΔB;L288R. Scale bars: 5 µm. d Quantification of percent UTs colocalized with SMARCAL1 in (c). Mean of three independent experiments ± SD from a minimum 300 nuclei analyzed per experiment. **P = 0.0032 by one-way ANOVA. ns non-significant. e In-gel single-strand G overhang (native, left panel) and total telomere (denatured, right panel) analysis in Rap1^–/– MEFs expressing the indicated cDNA constructs at the indicated time point with ±T7 endonuclease I (T7 Endo I) and Exo I treatment. Single-strand G overhang and total telomere signals in vector was set at 100% after normalizing each lane with ethidium bromide staining that served as an internal loading control. The numbers below the gel represents single-strand G overhang and total telomere signals. Molecular weights are displayed on the right. f Quantification of normalized telomeric DNA trapped in the well (outlined by the box) at indicated time point in (e) from one representative experiment (see also Supplementary Fig. 4f). Signal intensities in vector was set at 100% after normalizing with sub telomeres signals that served as an internal loading control. g T-complex and T-circle analysis using two-dimensional (2D) gel electrophoresis from genomic DNA isolated from Rap1^–/– MEFs expressing the indicated DNA constructs in ±0.5 µM APH treated with 40 units of T7 endonuclease I. T-complex (outlined by the box), t-circle (green arrowheads), ss G (red arrowheads), ds TRF (blue arrowheads). h Quantification of T-complex in (g). T-complex signal intensities in vector was set at 100% after normalizing with sub telomeres signals that served as an internal loading control. The graph represents the mean ± SD from two independent experiments. *P = 0.0175 by two tailed unpaired t test. Source data are provided as a Source Data file.