Fig 2: PTCH1 protein is up-regulated in the airway epithelium of patients with COPD compared to subjects without COPD. Paraffin-embedded human kidney tissues stained with (A) secondary only and (B) PTCH1 antibody were shown. Paraffin-embedded human lung tissues from subjects (C) without COPD, (D) COPD GOLD STAGE 2, and (E) COPD GOLD STAGE 3 were stained with PTCH1 antibody. Airway epithelium-specific PTCH1 protein expression was normalized to the length of basement membrane (µm) in (F) non-COPD, COPD GOLD STAGE 2 and GOLD STAGE 3/4, and (G) with COPD stratified by smoking status (current vs ex-smokers). (H) PTCH1 mRNA expression was normalized to GAPDH and expressed as ∆∆ct in human bronchial brushings from subjects with or without COPD. (I) Airway epithelial thickness and (J) total airway epithelial cell counts were quantified in subjects without COPD, COPD GOLD STAGE 2 and GOLD STAGE 3/4. Values were expressed as mean ± SEM. Kruskal–Wallis test with Dunnett’s multiple comparisons test was used in panels F,G. A two-tailed unpaired parametric t test was used in panel H. One-way analysis of variance was used in panels I,J. Correlations between total epithelial-specific PTCH1 protein expression (data log-transformed) with (K) epithelial thickness and (L) total epithelial cell count were shown. Linear regression analyses were used in panels K and L. Red dot = non-COPD, blue dot = COPD GOLD STAGE 2, orange dot = COLD GOLD STAGE 3/4. Representative immunofluorescence images of lung tissues from a patient with COPD GOLD STAGE 2 stained with (M) PTCH1 and MUC5AC (goblet cell) antibodies, (N) PTCH1 and FOXJ1 (ciliated cell) and (O) secondary only were shown.

Fig 3: ?Np63 is required for self-renewal of basal and distal lung stem cells.a–c Quantification of EdU incorporation in ?Np63fl/fl;RosaM/M and ?Np63?/?; Rosa?/? colonies at the indicated passages. d Quantification of Annexin V positive cells the same ?Np63fl/fl;RosaM/M and ?Np63?/?;Rosa?/? colonies shown in (a–d). Data were mean ± SD, n = 3, * vs. ?Np63fl/fl;RosaM/M, P < 0.05, two-tailed Student’s t-test. e Representative wells of tracheosphere formation assay of ?Np63fl/fl;RosaM/M and ?Np63?/?;Rosa?/? basal cells at passage 3. Scale bars equal 250 µm. f, g Quantification of number (f) and size (g) of tracheospheres formed by ?Np63fl/fl;RosaM/M and ?Np63?/?;Rosa?/? basal cells at the indicated passages. Data were mean ± SD, n = 3, * vs. ?Np63fl/fl;RosaM/M, P < 0.05, two-tailed Student’s t-test. h–s Representative images of tracheospheres in differentiation assay generated from ?Np63fl/fl;RosaM/M and ?Np63?/?;Rosa?/? basal cells, showing H&E stained cross-sections (h, i and n, o), and staining for Krt5/GFP (j and p), NGFR/GFP (k and q), Ac-tub/GFP (l and r) and Muc5ac/GFP (m and s). t Representative images of colonies formed by ?Np63fl/fl;RosaM/M and ?Np63?/?;Rosa?/? AT2 cells and BASCs. Scale bars equal 50 µm. u Quantification of the same colonies as in (t). Data were mean ± SD, n = 3, * vs. ?Np63fl/fl;RosaM/M, P < 0.05, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig 4: Mucin secretion defects in Syt2-mutant mice.a, Transverse sections of bronchial airways of mice stained with periodic acid fluorescent Schiff (PAFS) to demonstrate mucin with red fluorescence. Top, in naive mice without airway inflammation, scant intracellular mucin is visible. Treatment with IL-13 increases mucin synthesis, resulting in abundant intracellular mucin. Bottom, subsequent treatment with ATP induces mucin secretion, reducing intracellular mucin in WT C57Bl/6J mice (WT) and Syt2F/F mice (F/F), but not in Syt2D/D mice (D/D). Scale bar, 50 µm. b, Fractional mucin secretion was measured by analysing images of airways of mice treated with IL-13 alone and comparing those with those of mice treated with IL-13 followed by ATP, as shown in a. Individual data points and box plots are shown for two independent sets of experiments combined to give a total n mice (indicated below each box plot) per group (Supplementary Table 1). Comparison with the Syt2F/F group of mice was performed using two-tailed unpaired Student’s t-tests; ***P = 0.00026. c, Transverse sections of bronchial airways of mice treated with IL-13, then with methacholine (Mch) to induce smooth muscle contraction and mucin secretion, and fixed with methacarn and stained with PAFS to demonstrate lumenal mucus and residual intracellular mucin. Scale bar, 50 µm. d, The sum of the lumenal mucus cross-sectional area in the left lung measured at 500 µm intervals. Individual data points and box plots are shown for two independent sets of experiments combined to give a total of n mice (indicated below each box plot) per group (Supplementary Table 1). Comparison with the Syt2F/F group of mice was performed using two-tailed unpaired Student’s t-tests; **P = 0.0012.Source data

Fig 5: SP9 inhibits mucin secretion from airway epithelial cells.a, The design of synthesized SP9 with biotin or CPPs conjugated to the N terminus and Cy3 to the C terminus, respectively. Biotin–SP9–Cy3 was bound to streptavidin-conjugated C2 or CRM197. b, Diagram of the analysis of cumulated Cy3 intensities within individual HAE cells. c, Confocal collapse (projected) images of fixed HAE cells that were treated with SP9–Cy3 or SP9–Cy3 conjugated to bacterial toxins (C2, CRM197) or CPPs. Scale bar, 10 µm. The experiment was independently repeated twice with air–liquid interface (ALI) cultures from different donors with similar results. d, Quantitative analysis of intracellular Cy3 fluorescence for each peptide in MUC5AC+ HAE cells. Box plots and data points are shown for n cells (indicated below each box plot). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test; ***P < 0.0001. e, Schematic of the peptide application and sample collection from HAE cells maintained under ALI conditions. f, Representative western blot immunofluorescence images for MUC5AC on an apical surface of untreated HAE cells (control), HAE cells treated with 10 µM of SP9–Cy3, or 10 µM of either SP9–Cy3 or P9–Cy3 conjugated to CPPs for 30 min before stimulation. Basl., MUC5AC secretion during a 30 min period before stimulation (baseline). Exp., MUC5AC secreted within a 30 min experimental period with (IL-13 + ATP) or without (IL-13) stimulation of HAE cells with 100 µM ATP. Cells were treated with IL-13 to induce mucous metaplasia. All of the original blots are shown in Supplementary Fig. 1a. g, h, The ratio of baseline to reference wash secretion (fold increase in baseline secretion over reference secretion) (g) and the ratio of experimental to baseline (fold increase in stimulated secretion over baseline secretion) (h) for each condition in f. The numbers below the box plots indicate n for each condition, representing individual ALI cultures derived from four donors for each condition. Statistical analysis was performed using two-way ANOVA followed by post hoc Dunnett’s test; *P = 0.013.Source data