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. 2021 May 18;12:2910. doi: 10.1038/s41467-021-22899-2

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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. 1 — a Quantitative analysis of total levels of FH-tagged RIF1, measured by intra-cellular FACS staining. Anti-HA mouse ascites 16B12 was used to stain the indicated cell lines. Rif1^FH/FH: homozygous knock-in FH-tagged RIF1, as a control of quantitative staining. The plot shows distributions of densities from HA signal, measured in arbitrary units. One representative experiment is shown. b Quantification from Fig. 1a. The bar plot represents the median intensities for the experiment shown and the error bars indicate 95% confidence intervals, calculated through bootstrapping with 10,000 iterations. c Total proteins were extracted from cells expressing untagged wild type RIF1 (WT), or from hemizygote cells expressing RIF1-TgWT or RIF1-ΔPP1 (HA tagged), and immunoprecipitated with anti-HA antibody. The input, immunoprecipitated complex and flow through were analysed by western blot with anti-mouse RIF1 affinity-purified rabbit polyclonal antibody (1240) and anti-PP1α. d Quantitative analysis of the levels of chromatin-associated FH-tagged RIF1 throughout the cell cycle measured by FACS staining. Cytoplasmic and nucleoplasmic proteins were pre-extracted before fixing chromatin-associated proteins. Anti-HA mouse ascites 16B12 was used to visualise FH-tagged RIF1 as in a. Cell cycle stage was determined by DNA quantification (DAPI staining). Means from the results from two independent experiments, each with 2 or 3 independent cell lines per genotype are summarised. The error bars indicate standard deviations. P values were calculated by two-sided unpaired t test. e Cell cycle distribution of the indicated cell lines, as determined by FACS quantification of EdU incorporation (S-phase) and DAPI staining (DNA amount). The mean value of three independent clones per genotype, from three experiments is shown. Error bars indicate the standard error of the mean. P values are calculated using two-sided Wilcoxon rank-sum test.