Fig. 2. Wound fibroblasts exhibit a distinct transcriptional signature.

a Gating strategy for skin fibroblast isolation. b Quantification of fibroblast frequency relative to live cells in unwounded (normal) skin (NS) and in wounds of WT and Act mice. n = 6, 6, 9, 3 for WT, n = 6, 7, 9, 3 for Act, for NS, 3-, 5-, or 7-day wounds, respectively. c Left: Venn diagram showing the top 100 most highly expressed (by RPKM) genes in NS and 5-day wound fibroblasts. Right: Cell line enrichment analysis of the 77 overlapping genes via ARCHS4 (by enrichR), showing top five cell lines with enrichment of these genes; fibroblast-like cells are in bold. d Analysis of differentially expressed genes (DEGs). Left: Numbers of DEGs (false discovery rate (FDR) < 0.05) for all individual group comparisons. Right: Absolute expression of selected top-expressed (by RPKM) and top up-regulated (in 5dw vs NS comparisons) genes in the four groups. n = 3 for WT_NS, WT_5dw, Act_NS, n = 2 for Act_5dw. e Venn diagram showing the DEGs (FDR < 0.05) in all 5dw vs NS comparisons. Red circle shows the 556 genes shared between all comparisons. f 367 shared up-regulated DEGs from e (Log₂FC > 1) were subjected to functional enrichment analysis using enrichR. Top 10 Gene Ontology (GO) Biological Processes are shown, with FDR and percentage of input out of total pathway genes (% genes). g Shared DEGs from e were subjected to Ingenuity Pathway Analysis (IPA). Selected annotated functions are shown with Activation Z-scores and numbers of input genes enriched in the respective functions (# genes). Z-score > 0 (red): predicted activation of function; Z-score < 0 (blue): predicted inhibition of function; P value < 9.0E-09 for all shown functions. h The 5-day wound fibroblast signature was subjected to GSEA against gene sets from wound myofibroblasts. Normalized Enrichment Scores (NES) and FDR values are shown; NES > 1: positive enrichment (red), NES < −1: negative enrichment (blue); FDR are color coded based on statistical significance (green). Wound myofibroblast gene sets include genes up/down-regulated in ɑ-SMA⁺ myofibroblasts from 7-day small excisional wounds¹¹; genes up-regulated in ɑ-SMA⁺ myofibroblasts from 12- or 26-day large excisional wounds¹⁰ and CD29^high, CD29^low, or adipocyte precursor myofibroblasts from 5-day small excisional wounds¹³. Graphs show mean±SEM and P values; two-way ANOVA and Bonferroni’s multiple comparison post hoc tests (a). All n numbers indicate biological replicates. Source data are provided as a Source Data file (Fig. 2).