Fig 7. Increased cell proliferation rates in terminal bronchioles of Nkx2-1-Cre; Igf1r^fl/fl mice at different stages during repair after naphthalene challenge.

(A-B) Representative images of Ki67 immunostaining (brown arrows) in terminal bronchioles of Igf1r^fl/fl (+/+) (A) and Nkx2-1-Cre; Igf1r^fl/fl (Cre/+) (B) lungs at 3dN. Note the increased presence of Ki67⁺ in the epithelium of Cre/+ mutant mice with respect to +/+ controls. (C) Quantification of Ki67-based proliferation rates in both genotypes (See S7 Fig for representative images). Numbers in bars indicate mice analyzed. (D-G) Identification of proliferating cells by anti-BrdU immunostaining (green labeled nuclei) counterstained in red with an anti-Scgb1a1 antibody to mark club cells, allowing for differentiation of three groups of cells: total BrdU⁺ cells in the bronchiolar epithelium (including Scgb1a1⁺ cells) (green arrows), BrdU⁺/Scgb1a1⁺ double labeled cells (red arrows) and BrdU⁺ cells under the epithelial basal membrane (blue arrows) at 3dN (D-E) and 7dN (F-G) in control (+/+) (D, F) and mutant (Cre/+) (E, G) lungs. al, alveolus; tb, terminal bronchiole. Scale bars: 50 μm in A-B; 25 μm in D-G. (H) Quantification of BrdU⁺ cells in terminal bronchioles of control (+/+) and mutant (Cre/+) mice. The graph represents the quantification of total BrdU+ cells found in the epithelium (green bars), BrdU⁺/Scgb1a1⁺ club epithelial cells (red/pink bars) and in blue bars BrdU+ cells under the basal membrane (up to 12 μm distance) in non-treated (NT), and at three (3dN), seven (7dN) and fourteen (14dN) days after naphthalene treatment (red/pink bars) (see S8 Fig for representative images). Values in graphs show mean ± SEM. The statistical significance between stages is indicated by specified symbols as follows: *, +/Crevs. +/+ in same condition; @, vs. NT of same genotype; #, vs. 3dN of same genotype; $, vs. 24dN of same genotype; one symbol, p<0.05; two symbols, p<0.01; three symbols, p<0.001.