Extended Data Fig. 5. Characterization of Hh-activation during homeostasis and in fibrotic repair.

(a) RNAScope in situ of Shh shows expression in ectopic SOX2+ cells in the alveoli after bleomycin injury. This experiment was repeated independently twice with similar results.

(b,c) Histology quantification shows no evidence of KRT5+ metaplasia in Hh-activated animals without injury when compared to controls (n = 3 per group; each data point represents one animal; one-tailed unpaired Student’s t-test). Data are expressed as mean ± SD.

(d-g) After bleomycin injury, Hh-activated lungs show similar expansion of Gli1+ cells in distal alveoli compared to controls, a trend towards increased myofibroblasts (SMA+) differentiation of the Gli1 Lin+ cells in the alveoli, and no difference in ectopic SCGB1A1+ cells in the alveoli (for (e), n = 4 for control, n = 6 for Hh-activated; for (f,g), n = 9 per group; each data point represents one animal; one-tailed unpaired Student’s t-test for (e-g)). Data are expressed as mean ± SD.

(h) Model of 3D airway organoid assay using Hh-inducible mesenchyme whereby pretreatment of 4-hydroxytamoxifen (4OHT) induces Hh activation in the mesenchyme as shown by upregulation of Gli1 transcript compared to vehicle (ethanol) (n = 3 per group; each data point represents one biological replicate; one-tailed unpaired Student’s t-test).

(i-l) Mesenchymal Hh activation in vitro reduces CFE and organoid size, but increases the expression of Krt5 and number of KRT5+ organoids while reducing Sftpc expression and number of SFTPC+ organoids ((i-k) n = 3 per group; (l) n = 3 for Hh-activated, n = 5 for control; each data point represents one well; one-tailed unpaired Student’s t-test for (j-l)). Data are expressed as mean ± SD. Hh = hedgehog, 4OHT= 4-hydroxytamoxifen, AW = airway. Scale bars, 100 µm.