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. 2021 Nov 23;7:111. doi: 10.1038/s41421-021-00328-4

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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 Schematic diagram showing the dual recombinase-mediated sequential intersectional genetic strategy. Step 1: Dre-rox recombination switches CD11b-CrexER to the CD11b-Cre genotype in smMHC⁺ cells; Step 2: when CD11b is activated, Cre-loxP recombination genetically labels CD11b-expressing cells. b Flow cytometric analysis of tdTomato⁺ cells in the blood, spleen, and bone marrow of Myh11-Dre;CD11b-CrexER;R26-tdT;LDLR^−/− mice. c Schematic diagram showing the experimental strategy. Mice were treated with a high-fat diet (HFD) or normal laboratory diet. d Immunostaining for tdT and aSMA on aortic sections from 16-week-old mice treated with HFD or normal diet. e Whole-mount images of aortas after Sudan IV staining. f Immunostaining for tdTomato (tdT) and aSMA in aortic sections from mice treated with HFD or normal diet. g Immunostaining for tdT and CD45, CD11b, F4/80, LGALS3, or CD68 on plaque sections. Arrowheads, tdT⁺ macrophages. h Quantification of % of tdT⁺ cells expressing different macrophage markers in the plaques. Data are expressed as means ± SEM; n = 5. i Immunostaining for tdT and SMC markers. Arrowheads, tdT⁺ SMCs. j Quantification of the % of tdT⁺ cells expressing different SMC markers in the plaques. Data are expressed as means ± SEM; n = 5. k Immunostaining for tdT and fibroblast or pericyte markers. Arrowheads, tdT⁺ fibroblasts or pericytes. l Quantification of the % of tdT⁺ cells expressing fibroblast or pericyte markers in the plaques. Data are expressed as means ± SEM; n = 5. m Quantification of the % of fibroblasts or pericytes expressing tdT in the plaques. Data are expressed as means ± SEM; n = 5. n Flow cytometric analysis of different lineage markers expressed by tdT⁺ cells isolated from the plaques. o tSNE analysis of scRNA-seq data of 10,618 plaque cells from Myh11-Dre;CD11b-CrexER;R26-tdT;LDLR^−/− mice fed with 24 weeks HFD. p Feature plot of tdTomato in different clusters. 1946 tdT⁺ cells are presented in the clusters. q Cartoon image showing a subset of SMC-derived macrophage-like cells adopting a transient cell fate and contributing to SMCs, pericyte-like cells, and fibroblast-like cells in the fibrous cap and atherosclerotic plaques. Scale bars: black, 1 mm; white, 100 µm. Each image is a representative of five individual biological samples.