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. 2023 Mar 29;21:115. doi: 10.1186/s12916-023-02791-0

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© The Author(s) 2023

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Act1-knockdown in macrophages promoted epithelial-mesenchymal transition and CRC cell migration via activating CXCL9/10/11-CXCR3 and immunosuppression via PD1/PD-L1 axis. (A) CXCR3 expression was assayed by immunohistochemical staining in the tumor tissue of CRC patients, n = 6 (a), tumor of Apc^Min/+ and AA mice at 15^th week (b), and CT26 or MC38 cells cocultured with BMDMs of wild type and anti-Act1 mice for 48 h (c). Scale bar in (a), 200 μm (upper) and 50 μm (lower). Scale bar in (b), 100 μm. (B) The effect of cocultured macrophages on the migration of CXCR3-knockdown MC38 cells. Schematic representation of the experimental design (a). Expression levels of epithelial-mesenchymal transition markers in CXCR3 knockdown MC38 cells cocultured with wild type or anti-Act1 macrophages for 48 h (b). (C) The effect of CXCR3 knockdown in MC38 cells on macrophage-mediated migration of MC38 cells. (a) Transwell migration assay of MC38 cells after coculture with anti-Act1 macrophages for 24 h. Scale bar, 50 μm. (b) Wound-healing assay of MC38 cells during incubation with macrophages of anti-Act1 mice for 0 h and 24 h. Scale bar, 200 μm. (D) Immunofluorescence staining for PD-L1 (red) and F4/80 (green) (a), PD1 (green) and CD8⁺ T (red) (b) in AA mice at the 15^th week was taken by laser confocal microscope. Scale bars, 10 μm. (E) Immunofluorescence staining for PD-L1 (red) and CD68⁺ (green) (a), PD1 (green) and CD8⁺ T (red) (b) in CRC patients was taken by an immunofluorescent microscope. Scale bars, 10 μm. (F) The effect of anti-PD-L1 on the adenocarcinoma transition in AA mice after treatment 3 weeks. (a) Graphical depiction of anti-PD-L1 treatment. (b) Representative H&E staining of intestinal tumors of AA mice treated with PBS or anti-PD-L1. Scale bar, 100 μm. (c) Methylene blue staining of tumors in the small intestine from AA mice treated with PBS or anti-PD-L1 after 3 weeks. (d-e) Quantification of tumor number and tumor size in AA mice after 3 weeks of anti-PD-L1 treatment, n = 3. *p < 0.05, ^#p < 0.05, **p < 0.01, ^##p < 0.01, ***p < 0.001, and ****p < 0.0001 are considered statistically significant