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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 triggers R-loop induced replication stress in UtSMCs. (A) Temporal kinetics of replication stress marker activation in UtSMCs following Mediator kinase inhibition. UtSMCs were treated with CCT251545 (100 nM) for the indicated time points prior to immunocytochemical analysis with antibodies specific for R-loops (S9.6), activated replication stress signaling markers [pRPA (Ser33), pATR (Ser428), pCHK1 (Ser317), pCDC25A (Ser124)] and the DNA damage marker γH2AX. Data are plotted as fold-change in antibody signal intensity relative to untreated cells (0 h). Statistical significance was calculated using One-Way ANOVA followed Tukey’s Post hoc test, ****p ≤ 0.0001, ***p < 0.001; **p < 0.01. and *p ≤ 0.05. (B) Representative images of data plotted in (B). Images were captured at the peak time points for each marker. A parallel experiment was performed in UtSMCs overexpressing RNaseH (+ RNaseH). Campothecin (CPT; 10 μM) was included as positive control for R-loop induced replication stress signaling.