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. 2022 Apr 6;12:5827. doi: 10.1038/s41598-022-09614-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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MMR-deficient cells are resistant to the combination of TMZ and ATR inhibitor in clonogenic survival assay. LN229 cells were pre-treated with 12.5 μM of TMZ for 72 h before counting and seeding these cells into new plates with fresh media. Cells were seeded at various dilutions in triplicate (n = 3) before adding BAY-1895344 at a constant concentration of 2.5 nM for 14 days. Plates remained undisturbed in the incubator for 14 days before staining and counting colonies. (A) shMSH2, (B) shMSH6, (C) shMLH1, and (D) shPMS2 cells are resistant to TMZ and the combination with BAY-1895344. (E) shMSH3 cells are sensitive to TMZ and BAY-1895344. In all graphs, the solid black line indicates the wild-type LN229 MMR-proficient MGMT- cells treated with TMZ and the dashed black line indicates the same cells treated with TMZ and ATR inhibitor BAY-1895344. The solid gray line represents the shRNA MMR knockdown cell line treated with TMZ alone, and the dashed gray line represents the shRNA MMR knockdown cell line treated in combination with TMZ and BAY-1895344. Standard deviation was calculated from the triplicate values of colonies counted. Unpaired t-test was performed using GraphPad PRISM between LN229 MGMT- wild-type cells (dashed black line) and the shMMR cells (dashed grey line) for the TMZ + BAY-1895344 combination (****p < 0.0001).