Fig 2: Characteristics of mesenchymal fibroblasts in fibrotic skin disease.a Feature plots of the expression distribution for POSTN and COL11A1 in keloid fibroblasts (KF) and normal scar fibroblasts (NS). Expression levels for each cell are color-coded and overlaid onto the UMAP plot. The average cell expression of each gene in mesenchymal fibroblasts between KF and NS were shown in the right panel (Two-sided unpaired t-test). n = 3 biologically independent samples. In box plots, lines in the middle of boxes correspond to median values. Lower and upper hinges correspond to the first and third quartiles, and the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR (inter-quartile range) from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. b Heatmap of differentially expressed genes in each fibroblast subpopulations. MF: mesenchymal fibroblast, PIF: pro-inflammatory fibroblast, SRF: secretory-reticular fibroblast, SPF: secretory-papillary fibroblast. c Violin plots illustrating the expression of some marker genes in each fibroblast subpopulations. d Functional enrichment of upregulated genes in mesenchymal fibroblasts [Fisher exact-test, corrected for multiple comparisons using Benjamini and Hochberg method, adjusted P-value of <0.05]. e The top 13 candidate master regulators for mesenchymal fibroblasts identified by an algorithm for master regulator analysis algorithm (MARINa). Violin plots showing the relative expression levels of each master regulator in the right panel. f Immunofluorescence staining of NREP and ADAM12 in normal and keloid tissues. Lower panels are the insets of upper panels. Arrowheads indicate NREP+/ADAM12+ cells. Scale bar = 200 µm. g Percentage of NREP+/ADAM12+ cells in normal and keloid tissues. Data are presented as mean values ± SD (n = 5 images examined over 3 independent experiments). Two-sided unpaired t-test, ***P = 0.00013.

Fig 3: The supernatant of mesenchymal fibroblasts promotes collagen expression in keloid fibroblasts.a Isolation of CD266+CD9- and other fibroblasts from keloid dermis by flow cytometry. b qRT-PCR assay of some mesenchymal fibroblast marker genes expression in CD266+/CD9- fibroblasts and other fibroblasts from keloid dermis. Data are presented as mean values ± SD (n = 3 biologically independent experiments.). Two-sided unpaired t-test, ***P < 0.001(ASPN:P = 0.00003, COL11A1:P = 0.00019, POSTN:P = 0.00004, ADAM12:P = 0.00015, P311:P = 0.00019, COMP:P = 0.00002). c Western blot assay of mesenchymal fibroblast marker genes expression in CD266+/CD9- fibroblasts and other fibroblasts from keloid dermis. The experiments were repeated three times with three different fibroblast donors, and here a representative result was shown. d GO Biological Process enrichment analysis of differentially expressed genes between keloid CD266+/CD9- fibroblasts and other fibroblasts. e GSEA enrichment plots for representative signaling pathways upregulated in keloid CD266+/CD9- fibroblasts compared to other fibroblasts. (NES normalized enrichment score, corrected for multiple comparisons using FDR method, P-value were showed in plots). f, g Keloid other fibroblasts were treated with the supernatant of CD266+/CD9- or other fibroblasts. The expression of collagen I and collagen III was analyzed by qRT-PCR (f) or western blot (g). Data are presented as mean values ± SD (n = 3 biologically independent experiments). Two-sided unpaired t-test, **P < 0.01, ***P < 0.001. (COL1A1: P = 0.000009 and P = 0.000015, respectively; COL3A1: P = 0.00006 and P = 0.00214, respectively). The experiments were repeated three times with three different fibroblast donors, and here a representative result was shown. h Keloid other fibroblasts were treated as indicated in the figure. The expression of collagen I and collagen III was analyzed by western blot. The POSTN antibody neutralizing experiments were repeated three times with three different fibroblast donors, and here a representative result was shown.