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. 2019 Sep 10;47(20):10678–10692. doi: 10.1093/nar/gkz784

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© The Author(s) 2019. 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 Non-Commercial License (http://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 7. — CSB phosphorylation on S1276 promotes efficient recruitment of BRCA1-C to DSBs. (A) Quantification of the intensity of BRCA1 signal at the site of FokI-induced DSBs in U2OS-265-CSB-KO cells expressing various Myc-CSB alleles as indicated. The respective numbers of cells analyzed for the vector alone, Myc-CSB WT and Myc-CSB-S1267A were 247, 239 and 249. Data are represented as scatter plot graphs from single experiments with the mean indicated in this panel, B, C and H. The P value was determined using non-parametric Mann-Whitney rank-sum t-test in this panel, B, C and H. (B) Quantification of the intensity of MRE11 signal at the site of FokI-induced DSBs in U2OS-265 CSB-KO cells expressing various Myc-CSB alleles as indicated. The respective numbers of cells analyzed for the vector alone, Myc-CSB WT and Myc-CSB-S1267A were 221, 215 and 217. (C) Quantification of the intensity of CtIP signal at the site of FokI-induced DSBs in U2OS-265 CSB-KO cells expressing various Myc-CSB alleles as indicated. The respective numbers of cells analyzed for the vector alone, Myc-CSB WT and Myc-CSB-S1267A were 213, 212 and 232. (D) Quantification of cells with ≥ 10 IR-induced BRCA1 foci. hTERT-RPE-CSB-KO cells expressing various Myc-CSB alleles as indicated were treated with 2 Gy IR and fixed 1 h later in this panel, 7E and 7I. At least 500 cells were scored per condition in a blind manner in this panel, 7E and 7I. Standard deviations from three independent experiments are indicated in this panel, 7E-7G, 7I and 7J. (E) Quantification of cells with ≥ 10 IR-induced MRE11 foci. (F) Quantification of the percentage of synchronized mCherry-LacR-CSB- or mCherry-LacR-CSB-S1276A-expressing U2OS-265 cells with MRE11 at the lac operator array. At least 200 mCherry-positive cells were scored per condition in a blind manner in this panel, 7G and 7J. DTB: double thymidine block. (G) Quantification of the percentage of synchronized mCherry-LacR-CSB- or mCherry-LacR-CSB-S1276A-expressing U2OS-265 cells with BRCA1 at the lac operator array. (H) Quantification of the intensity of RIF1 signal at the site of FokI-induced DSBs in U2OS-265 CSB KO cells expressing various Myc-CSB alleles as indicated. The respective numbers of cells analyzed for the vector alone, Myc-CSB WT and Myc-CSB-S1267A were 218, 245 and 232. (I) Quantification of cyclin A-positive cells with ≥10 IR-induced RIF1 foci. (J) Quantification of the percentage of synchronized mCherry-LacR-CSB- or mCherry-LacR-CSB-S1276A-expressing U2OS-265 cells with RIF1 at the lac operator array.