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. 2022 Jul 16;13:4143. doi: 10.1038/s41467-022-31865-5

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© The Author(s) 2022

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. 5 — a Confocal micrographs (maximum intensity projections) of Nocodazole-arrested RPE-1 wild type, MDC1 knock-out (ΔMDC1) and CIP2A knock-out (ΔCIP2A) cells, irradiated with 1 Gy and stained for TOPBP1 and CIP2A. b Quantification of the experiment in a. CIP2A/TOPBP1 foci were manually counted. Each data point represents one mitotic cell (no IR WT: n = 52, ΔMDC1: n = 56, ΔCIP2A: n = 51, IR WT: n = 80, ΔMDC1: n = 61, ΔCIP2A: n = 53, pooled from three independent experiments) and bars represent the median. Statistical significance was assessed with the Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). c Flag-immunoprecipitation from 293FT cells transfected with Flag-tagged full-length CIP2A and HA-tagged MDC1 wild type (WT) and Ser168/Ser196 double mutant (S168A/S196A). d Quantification of CIP2A-MDC1 proximity by in situ PLA in U2OS wild type (WT) and U2OS MDC1 knock-out cells (ΔMDC1), arrested in mitosis by Nocodazole and mock treated or treated with 1 Gy of IR. Each data point represents one cell (WT -IR: n = 131, WT + IR: n = 114, ΔMDC1 -IR: n = 85, ΔMDC1 + IR n = 54, pooled from two independent experiments), and bars represent median. Statistical significance was assessed by Kruskal-Wallis test and Dunn’s multiple comparison test (α = 0.05). e Confocal micrographs (maximum intensity projections) of Nocodazole-arrested U2OS ΔMDC1 cells stably transfected with GFP-tagged wild type and S168A/S196A mutated MDC1, stained for CIP2A and TOPBP1 1 h treatment with 1 Gy of IR. f Quantitative analysis of GFP-MDC1 and CIP2A co-localization by SQUASSH: Left graph: object size colocalization (area of object overlap divided by total object area). Right graph: object number colocalization (fraction of objects in each channel that overlap ≥ 50%). Data points represent individual mitotic cells (n = 24, except S168A/196 A object number: n = 21, pooled from three independent experiments). Bars and error bars represent mean and SD. Statistical significance was assessed by unpaired t-tests with Welch’s correction (α = 0.05). Source data are provided as a Source Data file.