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. 2020 Jan 29;10:1443. doi: 10.1038/s41598-020-58084-6

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© The Author(s) 2020

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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Real time recruitment of 53BP1-GFP in U2OS cells after irradiation with 750 MeV/u uranium ions shows also individually delayed off-track foci. (A) U2OS nuclei expressing 53BP1-GFP showing accumulation at DSBs both at ion trajectories as well as off-track. (B) Nuclear wide recruitment kinetics of 53BP1 to early occurring DNA DSBs induced by 750 MeV/u U ions is characterized by a more sigmoidal recruitment behavior with a slower accumulation compared to NBS1 (see Fig. 2). Also for 53BP1, the protein accumulation at off-track DSBs appears more transiently (significant for t > 20 min, two-sided t-test p = 0.05) compared to the accumulation at the track with a maximum around 15–20 min post-irradiation, most probably indicating faster repair due to lower complexity. (n = 28 nuclei). Error bars 95% CI. (C) Selected traces of 53BP1-GFP recruitment to individual off-track foci showing clearly delayed onset of responses for some off-track DSBs. To better compare the kinetic behaviour, the clearly delayed recruiting DSBs were omitted in the global analysis of B. (D) Analysis of lag phases of off-track 53BP1 foci formation showed a broad distribution with a median at 465 s post-irradiation in comparison to 240 s for in track foci. (n = 674 or 274 RIF, respectively). The difference of the distributions is highly significant (Mann-Whitney U-test). Image montage of (A) was done using ImageJ 1.48 v (https://imagej.nih.gov). Graphs were generated using Origin Pro V.2019 (Originlab Corp., Northampton, MA, USA).