Fig. 1

Mucus hyperproduction in the bronchiolar epithelium of Adgrf5^−/− mice. a–d Alcian blue staining was performed on lung sections from WT (a and c) and Adgrf5^−/− mice (b and d) at 10 (a and b) and 30 (c and d) weeks of age. Mucus was stained blue. Nuclei (blue purple) and cytoplasm (pink) were counter-stained by hematoxylin and eosin. Insets show high magnification images of areas indicated by squares. Scale bars, 50 μm. e The volume of Alcian blue-positive mucosubstance in the epithelium was estimated from more than six randomly-selected bronchioles per mouse. Results are expressed as the volume of mucosubstance (nl) per surface area (mm²) of the bronchiole basement membrane, and are presented as the mean ± SEM (n = 5). **p < 0.01 and ***p < 0.0005. f The density of Alcian blue (AB)-positive cells was evaluated in more than six randomly selected bronchioles per mouse. Results are expressed as the number of Alcian blue-positive cells per millimeter length of bronchial basement membrane (BM), and are presented as mean ± SEM (n = 5). ***p < 0.0005. g–j The mRNA expression of Muc5ac (g), Muc5b (h), Slc26a4 (i), and Clca1 (j) was analyzed by qPCR in whole (1 week old) or post-lavage (3, 6, 8, 12, and 30 weeks of age) lungs from WT and Adgrf5^−/− mice. The data were normalized to Gapdh levels and expressed as values relative to corresponding WT mice. Values are presented as the mean ± SEM (n = 3–4). *p < 0.05; **p < 0.01; ***p < 0.001. k and l Immunofluorescence confocal microscopy was performed on lung sections from Adgrf5^−/− (k) and WT mice (l) at 30 weeks of age with an antibody specific for MUC5AC (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars, 40 μm