Fig. 4

Subepithelial fibrosis in the lungs of aged Adgrf5^−/− mice. a–d and g Masson’s trichrome staining was performed on lung sections from WT (a and c) and Adgrf5^−/− mice (b, d, and g) at 10 (a and b) and 30 (c, d, and g) weeks of age. Fibrous collagen was stained blue, and nuclei and cytoplasm were stained red. Br, bronchiole and V, blood vessel. Scale bars, 100 μm. e Trichrome-stained areas were evaluated in more than six randomly-selected bronchioles per mouse. Results are expressed as the area of trichrome staining (pixels) per micrometer length of bronchiole basement membrane, and are presented as the mean ± SEM (n = 3). **p < 0.01 and ***p < 0.001. f The amounts of fibrillar collagens and non-collagenous proteins in lung sections from WT and Adgrf5^−/− mice (4, 10, and 30 weeks of age) were estimated using a Sirius red/Fast green collagen staining kit. Results are expressed as the amount of fibrillar collagens (ng) relative to non-collagenous protein levels (μg), and are presented as mean ± SEM (n = 3). *p < 0.05 and **p < 0.01. h–k The mRNA expression of Col1a1 (h), Fn1 (i), Tnc (j), and Tgfb1 (k) 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. l Whole lung lysates (20 μg of protein) from WT and Adgrf5^−/−mice (KO) (3, 5, 12, and 30 weeks of age) were analyzed by western blotting using antibodies specific for TGF-β1 (top) and α-tubulin (bottom)