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. 2017 Nov 1;7:14882. doi: 10.1038/s41598-017-14922-8

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© The Author(s) 2017

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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Comparison of the R-G IF for SMLM images of U2OS cell nuclei labeled for proteins involved in NHEJ and HR (a) A schematic of the experimental timeline for NCS treatment and fixation of U2OS cells quantified in part b&c (left) and part d (right). (b, i) pDNA-PKcs and LigIV. Examples of an area of SMLM images of U2OS cells in control (left) and after NCS treatment (DNA-damaged; right) labeled for LigIV (green; outlined magenta: segmented clusters) and pDNA-PKcs (blue; outlined yellow: segmented clusters). Scale bar = 400 nm. (b, ii) Area of overlaps between pDNA-PKcs/LigIV was greater in DNA-damaged compared to control group. (b, iii) Number of overlaps between pDNA-PKcs/LigIV was greater in DNA-damaged compared to control group. (b, iv) Percentage of LigIV clusters overlapping with pDNA-PKcs was greater in DNA-damaged compared to control group. (b, v) IF between pDNA-PKcs/LigIV was greater in DNA-damaged compared to control group. Groups: control (n = 21); DNA-damaged (n = 29). (c, i) pATM and LigIV. Examples of an area of SMLM images of U2OS cells in control (left) and after NCS treatment (DNA-damaged; right) labeled for LigIV (green; outlined magenta: segmented clusters) and pATM (blue; outlined yellow: segmented clusters). Scale bar = 400 nm. (c, ii) Area of overlaps between pATM/LigIV was greater in DNA-damaged compared to control group. (c, iii) Number of overlaps between pATM/LigIV was greater in DNA-damaged compared to control group. (c, iv) Percentage of LigIV clusters overlapping with pATM was greater in DNA-damaged compared to control group. (c, v) IF between pATM/LigIV was greater in DNA-damaged compared to control group. Groups: control (n = 25); DNA-damaged (n = 13). (d, i) BRCA1 and BRIP1. Examples of an area of SMLM images of U2OS cells in control (left) and after NCS treatment (DNA-damaged; right) labeled for BRIP1 (green; outlined magenta: segmented clusters) and BRCA1 (magenta; outlined white: segmented clusters). Scale bar = 400 nm. (d, ii) Area of overlaps between BRCA1/BRIP1 was greater in DNA-damaged compared to control group. (d, iii) Number of overlaps between BRCA1/BRIP1 was greater in DNA-damaged compared control group. (d, iv) Percentage of BRIP1 clusters overlapping with BRCA1 was greater in DNA-damaged compared to control group. (d, v) IF between BRCA1/BRIP1 was not significantly different after DNA-damage compared to control group (n.s. = not significant; p > 0.05). Groups: control (n = 17); DNA-damaged (n = 18). (b–d) Error bars represent 95 CI. *p < 0.05, ***p < 0.001 ****p < 0.0001 by t-test.