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. 2021 Feb 12;12:1003. doi: 10.1038/s41467-021-21162-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 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. 1 — a Immunofluorescence staining of Caco-2 cells showing γH2AX (white). Phalloidin (red) stains for actin filaments and Hoechst (blue) stains for DNA. Caco-2 cells infected with pks+ WT E. coli are γH2AX-positive, display megalocytosis, and are multinucleated. Less γH2AX is detected in cells infected with the mutant strain (E. coli ΔclbR) defective for colibactin synthesis. Image representative of three independent replicates. Scale bars: 10 μm. b Epithelial cells from colon organoids are sheared and seeded on collagen-coated polycarbonate filter inserts. The cells attach and form a columnar polarized monolayer. Secreted mucus accumulates on the apical side. c Top: Cross-section of the polarized monolayer showing expression of colonic mucus MUC2 (red) and β-catenin (green). Hoechst (blue) stains for DNA. Bottom: Cross-section of the polarized monolayer showing expression of basolateral E-cadherin (green) and the proliferation marker Ki67 (red). Hoechst (blue) stains for DNA. Image representative of three independent replicates. Scale bars: 10 μm. d tdTomato (red) expressing colonic monolayer immunostained for γH2AX (white); Hoechst (blue) stains for DNA. Megalocytosis, multinucleated cells, and γH2AX-positive cells are observed in monolayer infected with pks+ WT E. coli. Image representative of three independent replicates. Scale bars: 10 μm. e Murine colon organoids expressing membrane tdTomato (red) immunostained for γH2AX (white); Hoechst (blue) was used to stain for DNA. Cells in colon organoids are γH2AX-positive only when infected with the pks+ WT E. coli. Image representative of three independent replicates. Scale bars: 10 μm. f CFUs per 100,000 cells of murine colon organoids from three independent replicates. Data represent mean ± SD, p < 0.05, as calculated by two-sided unpaired Student’s t-test. g Comet assay of murine colon organoid cells (each data point represents the mean of >80 cells) from three independent replicates shows more DNA damage in pks+ WT E. coli-infected cells, which is comparable to etoposide, the positive control. Data represent mean ± SD, p < 0.05, as calculated by one-way ANOVA and Tukey’s test. Source data are provided as a Source Data file.