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. 2020 Jul 30;142(15):1448–1463. doi: 10.1161/CIRCULATIONAHA.119.045115

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© 2020 The Authors.

Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 4. — Distinguishing features of fibrosis-associated fibroblasts.

A, GO terms enriched in a set of genes derived from comparing gene expression differences between Fibroblast-Cilp and Fibroblast-Thbs4 vs all other control cells. x axis indicates the number of genes mapped to each GO term, and color indicates the adjusted P value from GO enrichment analysis. Outline of FIt-SNE projection shown on Figure 2D, Left, indicates Fibroblast-Cilp and Fibroblast-Thbs4 population (red) being compared with other cells (black; see Tables V and VI in the Data Supplement). B, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp and Fibroblast-Thbs4 vs fibroblasts from control mice. C, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp and Fibroblast-Thbs4 vs fibroblasts from AngII-treated mice. Purple FIt-SNE outline indicates AngII-fibroblasts being compared with Fibroblast-Cilp and Fibroblast-Thbs4. D, Presentation of GO terms as in A, but associated with a comparison of Fibroblast-Cilp to Fibroblast-Thbs4. E, Dot plot showing expression of fibroblast genes classified within the GO categories shown on y axis. Circle size represents the sum of transcripts for genes corresponding to each GO term within each cell population. See Figure XII in the Data Supplement for transcript levels of all cell types. F, Top 10 genes enriched for specific expression in either Fibroblast-Cilp (dark blue) or Fibroblast-Thbs4 (orange) cell populations. Columns show average expression in each cell population expressed as either normalized read counts (in units of unique molecular identifiers per 10 000; columns 2–3) or percentage of cells with nonzero expression of the gene (columns 4–5). G, Micrograph showing AngII-treated mouse heart section stained with anti-THBS4 antibody, wheat-germ agglutinin (WGA; to identify cell boundaries and fibrosis), and DAPI (nuclei). Figure insets indicate regions without fibrosis (i and i′); dense THBS4+ fibrosis (ii) and perivascular fibrosis without THBS4 staining (iii and iii′). Region with discrete THBS4+ cells is also shown (ii′). Scale bar, 100 µm. Comparable staining was performed on >4 AngII-treated mouse hearts. See Figure XIII in the Data Supplement for control micrographs. H, FIt-SNE plot with cells colored according to Acta2 transcript abundance (red=high, gray=low). Red outline indicates Fibroblast-Cilp and Fibroblast-Thbs4 population. I, Micrograph labeled as in F, however with additional marker for ACTA2. Monochromatic images show intensity levels of individual fluorescence channels. Scale bar, 25 µm. Comparable staining was performed on >3 AngII-treated mouse hearts. ACTA2 indicates smooth muscle actin–expressing; Adj., adjusted; AngII, angiotensin II; DAPI, 4′,6-diamidino-2-phenylindole; FIt-SNE, fast Fourier transform–accelerated interpolation–based t-distributed stochastic neighbor embedding; GO, gene ontology; TGFbeta, transforming growth factor beta; and THBS4, thrombospondin 4.