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. 2021 Apr 16;12:2281. doi: 10.1038/s41467-021-22450-3

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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 Protocol for induction of AOM/DSS-induced CAC in mice. Il11-Egfp reporter mice were intraperitoneally injected with AOM on day 0, followed by repeated DSS administration. Colorectal cancer gradually develops ~30 days after AOM injection. Unless otherwise indicated, the following experiments used tumor and nontumor tissues collected on day 98–105 after AOM/DSS treatment. b Representative image of tumor and adjacent nontumor tissues in the mouse colon on day 77 after AOM injection. Colon sections were stained with hematoxylin & eosin (H&E) (n = 20 mice). Scale bar, 200 μm. c mRNA samples from tumor and nontumor tissues were analyzed using qPCR to determine the expression levels of the indicated genes. Results are mean ± SEM (n = 6 mice). Results are representative of two independent experiments. d–f From colonic tumor tissues of Il11-Egfp reporter mice, single-cell suspensions were prepared, and percentages of IL-11⁺ (EGFP⁺) cells were determined. Representative flow cytometry images are shown (d). Results are mean ± SEM (n = 7 mice). Cells were stained with the indicated antibodies, and marker expression on EGFP⁺ cells was analyzed by flow cytometry. A representative dot plot from three independent experiments shows the percentages of EGFP⁺ cells expressing each marker (n = 5 mice except for CD34 where n = 4 mice) (e). Percentages of EGFP⁺ cells among podoplanin⁺ cells (f). Results are mean ± SEM (n = 5 mice). g Nontumor and tumor tissue sections were stained with anti-GFP antibody (n = 4 mice). Scale bars, 100 μm. h–l Tumor sections were stained with anti-GFP along with anti-IL-11 (h) (n = 4 mice), anti-E-cadherin (j) (n = 4 mice), or anti-Ki67 (k) and anti-podoplanin (k) antibodies (n = 5 mice). The right-hand panels show enlarged images of white boxes from the left-hand panels. White arrowheads indicate EGFP⁺ cells expressing the respective markers shown in red. IL-11⁺ and EGFP⁺ cell numbers were calculated, and IL-11⁺/EGFP⁺ areas as percentages of the total area are shown (i). Ki67⁺ and EGFP⁺ cell numbers were calculated, and are shown as percentages of the total area (l). Results are mean ± SEM (n = 5 mice). Scale bars, 100 μm. Statistical significance was determined using the two-tailed unpaired Student’s t-test (c, d). Source data are provided as a Source Data file.