Fig 2: HH pathway inhibition induced AEC remodelling. A, Representative immunoblots from Shh immunoprecipitations in AEC culture medium (n = 3) with control IgGM (0.1 µg/ml and 1 µg/ml) and AB5E1 (0.1 µg/ml and 1 µg/ml). B, Schema depicting AEC cultures in the ALI model and AB5E1 treatment in culture medium to investigate transcript levels, protein levels and localizations, and TEER during the process of differentiation. C, Examples of micrographs taken from AEC cultures at ALI-14 showing basal cells (p63, green). Nuclei are stained in blue (DAPI). D, Box and whiskers plot (median with IQR) represents the percentage of p63+ cells (n = 5) on total cell population (evaluated on DAPI-stained cells count) at ALI-7 and ALI-14 in normal condition (black) and AB5E1-treated cells (red). *p < 0.05 CTL vs AB5E1. E, Examples of micrographs taken from AEC cultures at ALI-28 showing cilia (Arl13b, red). Nuclei are stained in blue (DAPI). F, Box and whiskers plot (median with IQR) represents the relative mean grey values of cilia-associated fluorescence (n = 8) at ALI-28 and ALI-35 (the baseline is given by the average value obtained from ALI-21 in normal condition) in normal condition (black) and AB5E1-treated cells (red). *p < 0.05 CTL vs AB5E1. G, Examples of micrographs taken from AEC cultures at ALI-21 showing mucins (Muc5b, red; Muc5ac, green). Nuclei are stained in blue (DAPI). H, Box and whiskers plot (median with IQR) represents the relative mean grey values of the mucins associated fluorescence ratios Muc5ac/Muc5b (n = 6) at ALI-21, ALI-28 and ALI-35 (the baseline is given by the average value obtained from ALI-14 in normal condition for each mucin) in normal condition (black) and AB5E1-treated cells (red). *p < 0.05 CTL vs AB5E1. I, Box and whiskers plots (median with IQR) represent the TEER of AEC (n = 5) at ALI-35 in control condition (black) or treated with AB5E1 (red). *p < 0.05 CTL vs AB5E1. J, Schema summarizing Shh inhibition impact on cell differentiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Gli2 expression is decreased in airway progenitor basal cell nuclei from COPD patients. a. Representative micrograph showing a ROI of a bronchial brushing stained for cilia (Acetylated tubulin, green); Gli2 (Gli2, red); basal cells (p63, white) and cell nuclei (DAPI, blue) in both non-COPD (upper panel) and COPD patients (lower panel). Magnification corresponding to the selected area is shown. Insets depict localization of the Gli2 transcription factor. b. Dot plot with median showing the percentage of Gli2-positive basal cell nuclei in non-COPD (n = 15) and COPD patients (n = 15). ***, p < 0.0001. c. Linear regression of the percentages of Gli2-positive basal cell nuclei according to FEV1 (% predicted) for non-COPD (n = 15) and COPD patients (n = 15). Non-COPD patients are represented by black circles and COPD patients are represented by red circles

Fig 4: Airway progenitor basal cell population is enriched in COPD. a. Study flow chart. b. Representative micrograph showing a Region of Interest (ROI) containing AEC obtained by bronchial brushing in a non-COPD patient stained for cilia (Arl13b, green); mucins (Muc5ac, red); basal cells (p63, white) and cell nuclei (DAPI, blue). Magnification corresponding to the selected area is shown. C. Dot plot with median showing the percentage of ciliated, goblet and basal cells in both non-COPD (n = 15) (black circle) and COPD patients (n = 15) (red circle). *, p < 0.05 and ***, p < 0.0001; non-COPD vs COPD

Fig 5: Human parechoviruses target p63 positive basal cells in the airway epithelium. Confocal image stacks of HPeV1- and HPeV3-infected HAE cultures triple-stained by (A) ciliated cell marker β-tubulin (red), basal cell marker p63 (purple) and HPeV antibody (green) or (B) secretory cell marker mucin 5B (red), basal cell marker p63 (purple) and HPeV antibody (green). Nuclei stained by DAPI (blue). Scale bars, 20 μm.