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. 2022 Apr 13;12:6169. doi: 10.1038/s41598-022-10188-x

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Mediator kinase inhibition alters replication fork dynamics and triggers S-phase delay in UtSM cells. (A) Representative images of combed DNA fibers from IdU/CldU pulse-labeled parental or RNAaseH overexpressing (−/ + RNaseH) UtSMCs cells treated with DMSO or CCT251545 (CCT; 100 nM). (B) Top, pulse labeling scheme. CCT251545 was added for 2 h between 30 min Idu (red) and CldU (green) pulses. Bottom, percentage of singly labeled fibers retaining only first (Idu) or second (CldU) label indicative of stalled or re-started forks, respectively. (C) Velocity of fork progression as determined from CldU tract lengths (kb/min). (D) Percentage of bidirectional asymmetric forks, represented as percentage of forks relative to total number of forks in the field. (E) Unsynchronized parental or RNaseH overexpressing (−/ + RNaseH) UtSMCs treated with DMSO or CCT251545 (CCT; 100 nM) for 3 days were stained with propidium iodide and analyzed by flow cytometry. Percentage of cells in each cell cycle phase is plotted. Statistical significance was calculated using One-Way ANOVA followed Tukey’s Post hoc test, ****p ≤ 0.0001, ***p < 0.001; **p < 0.01.