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. 2018 Feb 22;9:764. doi: 10.1038/s41467-018-03096-0

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© The Author(s) 2018

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. 2 — Loss of checkpoint control in the presence of MS-275. a HCT116 cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Cell cycle analysis shown as mean ± SD (n = 3). Control cells have G1 54%, S 22%, G2 24%; HU leads to G1 27%, S 55%, G2 18%; MS-275 leads to G1 69%, S 9%, G2 22%; HU/MS-275 leads to G1 30%, S 44%, G2 26%. Statistical significance is displayed for G2 cells (one-way ANOVA, *P < 0.05). b Treatment with HU (1 mM) and/or MS-275 (2 µM) was carried out as indicated (16–28 h). Representative histograms of cell cycle analysis are shown (n = 3). c HCT116 cells were treated as in a. Immunofluorescence was performed using Tubulin antibody followed by Alexa Fluor-488-coupled secondary antibody; TO-PRO3, nuclear staining. Top: representative pictures of treated cells; scale bar, 20 µm. Bottom: quantitative analysis of mitotic cells using Tubulin immunofluorescence. Data are per cent of mitotic cells relative to untreated cells (mean ± SD; n = 2; Student’s t-test; **P < 0.01). d DNA fibre assay: CldU (magenta tracks) and IdU (green tracks) were detected using specific primary and secondary antibodies. Replication tracks were classified according to schematic shown (left panel). Representative replication tracks are shown (right panel). e Quantitative analysis of replication tracks. Length was calculated in kb pairs/min (2.59 kb pairs = 1 µm). Data are mean ± SD (n = 3). f Cells were treated with 1 mM HU and 2 µM MS-275 for 6–24 h. Whole-cell extracts were blotted for WEE1, pCDK1 and HSP90 (loading control; n = 4). g Cells were treated as stated in f, immunoblot was done for Cyclin B and mSIN3A (loading control; n = 3). h Cells were treated as stated in f; immunoblot was performed for phosphorylated p53 (S15), p53, WIP1, p21 and Tubulin (loading control; n = 3). i Amounts of p53 and phosphorylated p53, with their levels in HU-treated cells set as 1. Data were normalised to loading controls. Results represent the mean ± SD (n = 4; one-way ANOVA; ***P < 0.001)