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. 2021 Jun 24;12:3937. doi: 10.1038/s41467-021-24224-3

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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. 1 — a Clonogenic survival of Tpr-depleted (siTPR) and control U-2 OS cells (siCTRL) treated with aphidicolin (Aph). Data are represented as mean ± SEM from three independent experiments (n = 3). Statistical analysis: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post analysis for multiple comparisons; ns not significant; *p value < 0.05. b Clonogenic survival of Tpr-depleted (siTPR) and control U-2 OS cells (siCTRL) treated with hydroxyurea (HU). Data are represented as mean ± SEM from three independent experiments (n = 3). Statistical analysis: Mann–Whitney test and Welch’s t-test; ns not significant; **p value < 0.01. c Representative images of 53BP1 foci in Tpr-depleted (siTPR) and control (siCTRL) U-2 OS cells. Cells were either nontreated (Mock) or treated with 0.2 μM aphidicolin for 48 h (Aph). The staining of cyclin A serves as a marker of the cell cycle stage. Nuclei were stained with DAPI. The scale bar, 7.5 µm. Top right: quantification of 53BP1 foci in Tpr-depleted (siTPR) and control U-2 OS cells (siCTRL) either nontreated (Mock) or exposed to 0.2 μM aphidicolin for 48 h (Aph). A series of nonoverlapping frames were randomly acquired by automated microscopy, and the 53BP1 foci in G1 nuclei (cyclin A negative) were counted. Data are represented as mean ± SD from three independent experiments (n = 3). Statistical analysis: Kruskal–Wallis test with Dunnett’s T3 post analysis for multiple comparisons. Bottom right: quantification of micronuclei in Tpr-depleted (siTPR) and control U-2 OS cells (siCTRL) performed on the same samples as 53BP1 foci analysis (top right panel). Data are represented as mean ± SEM from three independent experiments (n = 3). Statistical analysis: Kruskal–Wallis test with Dunnett’s T3 post analysis for multiple comparisons. d Depletion of Tpr leads to increased RPA pS4/8 phosphorylation upon hydroxyurea treatment. Cells were transfected with three independent siRNAs targeting Tpr (siTPR #53, siTPR #54, siTPR #55) or control siRNA (siCTRL). Transfected cells were either nontreated (Mock) or treated with 2 mM hydroxyurea (HU) for 4 h. Staining of RPA total is used as a loading control. Data are representative of three independent experiments. Source data for d are provided in the Source Data file.