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. 2021 Feb 11;11:3656. doi: 10.1038/s41598-021-83142-y

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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 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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Baseline DNA replication stress and cytosolic DNA in KRAS mutant (mut) vs wild-type (wt) cancer cells. (a) Illustration of DNA fibers in exponentially growing cells that were pulse-labeled with thymidine analogues CldU and IdU and lysed after which DNA fibers were spread and immunodetected with specific antibodies against CldU and IdU. (b, c) Frequency distributions of DNA fiber length (i.e., CldU tract length) in isogenic cell pairs. (d, e) Velocity of fork progression in a panel of cancer cell lines (H1703, Calu-6 – lung cancer; DLD1/DWT7 – colon cancer; U2OS – osteosarcoma; MDA-MB231 – breast cancer; FaDu – head/neck cancer) and in a control (Ctrl) normal fibroblast line. (f, g) Percentage of fibers retaining only the first label (CldU) indicating fork stalling in isogenic cell pairs. (h) Percentage of bidirectional asymmetric forks that progressed through replication fork arrest. (i) Levels of double-stranded (ds) cytosolic DNA using PicoGreen dsDNA staining on cytosolic fractions from untreated cells. (j) Left, representative images of untreated cells stained with a dsDNA-specific antibody or DAPI. Right, semiquantification of fluorescent signal. All bars represent mean + /- SEM based on at least 3 independent repeats. Statistical comparisons by two-sided T-test indicating *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.