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. 2021 Apr 9;4:451. doi: 10.1038/s42003-021-01979-5

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© The Author(s) 2021

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. 4 — Representative images of RPE1 mRuby-PCNA p21-GFP cells treated with DMSO (a) or CENPEi (b) for 16 h and washed 10 h prior to live-cell imaging for 14 h. Scale bar 25 μm. Yellow ± refers to p21 or PCNA foci positive or negative respectively. Images show a normal shaped nucleus with low p21 and nuclear PCNA foci and a multinucleate cell, exiting mitosis and building p21-GFP levels through time, without gaining nuclear PCNA foci. c Graph of the mean p21 level per nucleus for the normal, or per compartment for multinucleated cells from movies as in Fig. 4a & b. Values are normalised to the mean p21 value from the first time point measured. PCNA signal was used to identify nuclear areas. Colours of lines correspond to different nuclear compartments. d RPE1 cells treated with DMSO or CENPEi for 16 h were fixed 48 h later for immunostaining with antibodies against gamma H2AX and RNA pol II CTD pSer5. DNA was stained with DAPI. Representative images of non-overlap between gamma H2AX and RNA pol II pSer5 in cells following DMSO (control) or CENPEi treatment. Scale 15 μm; insets 5 μm. e Graph of the mean RNA pol II CTD pSer5 intensity per nucleus (normal or misshapen nuclei) or nuclear compartment (micronucleated and multinucleate). Each plot represents one nucleus/nuclear compartment respectively. N refers to the number of cells analysed, across 3 independent repeats. Statistical significance was assessed using a one way ANOVA with multiple comparisons. f Mean RNA pol II CTD pSer5 intensity per nucleus/nuclear compartments plotted against the size of the nucleus/nuclear compartment. Line colours represent nuclear morphology as indicated. N refers to the number of cells from 3 independent repeats.