Extended Data Fig. 4: Additional in vivo validation of early TGFβ-associated myofibroblasts, related to Fig. 2.

a-b, Nuclear flow cytometry showing elevated pSMAD3 expression within lesional fibroblasts (Pdgfrα^GFP-int) relative to dural/parenchymal fibroblasts (a) or other lesional cells (b), 7dpi. n = 4 mice. c-e, Image of αSMA⁺ expression among fibroblasts (Col1a2^creER; Rosa26^TdT+) at 7/21dpi (c), and quantification of the number (d) and percentage (e) of αSMA⁺ fibroblasts. n = 3 mice/group. f, Confocal microscopy showing Cthrc1^creER; Rosa26^TdT+ fibroblasts in whole-mounted dural meninges at indicated timepoints. Tamoxifen days ⁻16–⁻14 prior to injury (rest); days ⁻1–1 (2dpi); days 0–2,5 (7dpi); or days 0–2,5,12 (14dpi). g, Schematic showing tamoxifen induction for (h-l) and hypothesized trajectory for Acta2 (gene for αSMA) expression. h-l, Confocal microscopy showing Acta2-lineage⁺ lesional fibroblasts after PT injury (tamoxifen induction and harvest days indicated), with robust injury-induced Acta2 (TdT) expression (h) but lack of lineage-traced fibroblasts (i). Active Acta2 expression is reduced but present at 7dpi (j, shown at 14dpi) and absent by 14dpi (k, shown at 21dpi). l (inset from k) shows expected Acta2^creER; Rosa26^TdT+ vascular smooth muscle cells (white arrows) near CD31⁺ vessels. m-n, Image (m) and quantification (n) of fibroblast proliferation (EdU incorporation) 2 h after EdU pulse (“Pulse”; 7dpi, 14dpi, or 21dpi) or after EdU injection every other day (“Continuous”, and shown in m; 14dpi). In insets (m), arrows mark proliferating fibroblasts. Fibroblasts defined as Col1a2^creER; Rosa26^{TdT+/Sun1GFP+}. Tamoxifen days 0,1,2,5,12. n = 3 mice/timepoint (2 slices/mouse). o-p, Cthrc1-lineage⁺ fibroblasts within fibrotic scar after tMCAO (o) or TBI (p). Tamoxifen days 0,1,2,5,12 (o) or 0,1,2,5 (p). One-way repeated-measures ANOVA, Tukey post-test (a); paired two-way Student’s T-test (b); two-way Student’s T-test (d,e); one-way ANOVA, Tukey post-test (n). 14μm slices.

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