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. 2021 Oct 6;49(19):11083–11102. doi: 10.1093/nar/gkab892

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — USP39 is a key factor for maintenance of genomic integrity and cell survival. (A) The knockdown efficacy of each USP39 siRNA or its pool (siUSP39-A, -B and -C mixture) was tested in U2OS cells. (B) Ablation of endogenous USP39 leads to genomic instability. USP39-depletion induced genomic instability, which was monitored by a neutral comet assay (left panel), and the level of genomic fragmentation was quantified as indicated (right panel). siRNA-targeting LIG4 was used as a positive control for the comet assay. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test (C) Clonogenic cell survival assay. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. (D and E) Analysis of BRCA1 and 53BP1 IRIF formation. U2OS cells were transfected with control or USP39 siRNAs. Cells were fixed and stained with BRCA1 (D) or 53BP1 (E) antibodies, and nuclei were counterstained with DAPI as indicated by experimental conditions (left panel). Foci formation per cell ≥ 100 was calculated as indicated (right panel). Statistical significance was determined by the Student's t-test (F) Comparative analysis of HR and NHEJ repair activities in USP39 knockdown cells. siCtIP and siLIG4 were used as a positive control for HR and NHEJ repair assay, respectively. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test (G) GFP-^si^ReUSP39 was generated, and its resistance against USP39 siRNA was tested. (H) GFP-^si^ReUSP39 was reintroduced into cells with ablated endogenous USP39 and USP39-dependent regulation of genomic stability was monitored and quantified as described above. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test (I) USP39 is involved in the early response to DNA damage. USP39 siRNA and V5-^si^ReUSP39 were transfected into U2OS cells and 48 h later, cells were treated with γ-irradiation (2 Gy). The number of γH2AX foci at DSB sites was monitored (left panel) and quantified (right panel). Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. Data represent the mean ± s.e.m. from three hundred cells or more. All results represent at least three independent experiments. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. n.s., not significant.