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. 2021 Mar 26;4:417. doi: 10.1038/s42003-021-01941-5

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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. 3 — a Scheme of colony formation assays (CFAs) and change of p53 status with relative CENP-A protein levels over time for TetOn-CENPA-FLAG-HA cell lines. WT p53 is transiently activated in HeLa cells by a short heat shock, while p53 is stably inactivated in MCF10-2A cells by expression of a p53 dominant-negative peptide (p53-DN). b Temporary change of p53 status in HeLa cells by 1 h heat shock (HS). Western blot of HeLa cell TCEs at indicated time points following 1 h at 42 or 37 °C control. Temporary increase of p53 and subsequently p21. 1x load = ~33,000 cells. Primary antibodies are indicated on the right. ^# = high sensitivity ECL exposure. H4 used as loading control. c Increase of p53 levels in HeLa cells by heat shock. Western blot of TCEs after 1 h at 42 or 37 °C control. Loaded at 8x, 4x, 2x, 1x concentrations, where 1x load = ~12,500 cells. Primary antibodies are indicated on the right. ^# = high sensitivity ECL exposure. H4 used as loading control. Representative western blot from three independent experiments is shown. d HeLa S3 CFAs pertaining to scheme in (a). Each dot represents a single biological replicate. Plots show mean and 95% confidence interval. Statistical significance tested by two-tailed Welch’s t test with Bonferroni cutoff at a p value of 0.0125 (α = 0.05). * = p value < 0.01. e Change of p53 status in MCF10-2A TetOn-CENPA-FLAG-HA cells. Western blot of TCEs after 24 h of 10 µM Nutlin-3 treatment or DMSO control, as indicated. Dominant negative p53 peptide stabilizes p53 and suppresses its activation of p21. 1x load corresponds to total protein extract from ~30,000 cells. Primary antibodies are indicated on the right. ^# = high sensitivity ECL exposure. H4 used as loading control. Representative western blot from three independent experiments is shown. f MCF10-2A CFAs pertaining to scheme in (a). Each dot represents a single biological replicate. Plots show mean and 95% confidence interval. Statistical significance tested by two-tailed Welch’s t test with Bonferroni cutoff at a p value of 0.0125 (α = 0.05). *** = p value < 0.0001. See also Supplementary Fig. 1C, D for CFAs following range of Dox and X-irradiation doses and Supplementary Fig. 1E for representative CFA images.