Characterization of pulmonary remodelling in AAC-rats. (A) Immunofluorescent staining of frozen rat lung sections and confocal imaging with Click-iT 5-ethynyl-2′-deoxyuridine (EdU; white or pink nuclei = EdU-positive nuclei = proliferating cells) in combination with α-smooth-muscle actin (α-SMA; in green) in AAC-WT and AAC-Kcnk3-mutated rats. Counterstain was DAPI (blue). EdU positive nuclei are indicated by yellow/white arrows, and vessel lumen is indicated (v). Scale bar, 50 μm (left panels) and 20 μm (right panels). (B) Quantification of the percentage of lung proliferating cells (% of EdU-positive nuclei) in Sham-WT (n = 5), Sham-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 6), AAC-WT (n = 6), and AAC-Kcnk3^{Δ94Ex1/Δ94Ex1} rats (n = 6). (C) Quantification of the proportion of EdU positive + αSMA positive cells in in Sham-WT (n = 3), Sham-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 3), AAC-WT (n = 4), and AAC-Kcnk3^{Δ94Ex1/Δ94Ex1} rats (n = 6). (D) Upper panel, in a group of AAC-kcnk3-mutated rats alveolar wall appeared thickened. The alveolar septa enlargement is characterized by the affluence of inflammatory cells (mostly mononucleated), and at some degree to alveolar oedema. Lower panel, in severe cases the alveolar septa enlargement is hypercellular, with many inflammatory cells (red arrow), macrophages (blue arrow) and interstitial oedema (black star). Endoalveolar macrophages are also visible (dark blue arrows). No fibrin deposits nor alveolar necrosis are observed. Scale bars, 100 and 50 (zoom) μm. (E) Severe peri-bronchial/perivascular inflammation, extending in peri-bronchiolar alveoli. Scale bars, 100 and 25 (zoom) μm. (F) Analysis of the peri-bronchiolar inflammation score in Sham-WT (n = 4), Sham-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 4), AAC-WT (n = 4), and AAC-Kcnk3^{Δ94Ex1/Δ94Ex1} rats (n = 4). (G) Western blot images of the phosphorylation of STAT3 protein in lung lysates. (H) Quantification of the phosphorylation of STAT3 protein in lung from Sham-WT (n = 6), Sham-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 6), AAC-WT (n = 6), and AAC-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 7) rats (see the complete unedited gel in see Supplementary material online, Figure S8). (I) Relative mRNA expression (2^−ΔΔCt) of Il-6 in lung from Sham-WT (n = 6), Sham-Kcnk3^{Δ94Ex1/Δ94Ex1} (n = 6), AAC-WT (n = 4), and AAC-Kcnk3^{Δ94Ex1/Δ94Ex1} rats (n = 4). Experiments were normalized by 18S mRNA and analysed using ΔCt values. Data are represented as scatter dot plots, with mean ± SEM. Experiments presented in panels A–B, D–E, G–H, and I were analysed using two-way ANOVA followed by Sidak’s post hoc test, and experiments presented in panels C and F were analysed using one-way ANOVA followed by Dunn’s post hoc test after Kruskal–Wallis test,***P < 0.001. *P < 0.05, **P < 0.01, and ***P < 0.001. Edu⁺ and Edu⁺/αSMA⁺ and Stat3 phosphorylation were not normally distributed, and were log-transformed prior to statistical analysis.