Fig. 3
Lung phenotype of PC-specific Yap1,Wwtr1 mutants. a Maximum intensity projections of P7 and P12 Yap1,Wwtr1iPCKO and littermate control lungs showing NKX2.1-stained (green) AT1 and AT2 cell nuclei, LAMP3-stained AT2 cells (red), and DAPI (blue). Scale bar, 30 µm. b Quantitation of NKX2.1 positive and LAMP3 positive cells shown in a. Data represents mean ± s.e.m. (n = 4 mice; two-tailed unpaired t-test or Welch’s t-test). c Three dimensional high magnification reconstruction images of Yap1,Wwtr1iPCKO R26-mT/mG and littermate control lungs stained for GFP (white). Scale bar, 20 µm. d Flow cytometric analysis of PDGFRβ+ pericytes in P12 Yap1,Wwtr1iPCKO and control lungs. Data represents mean ± s.e.m. (n = 7 mice; NS not significant, two-tailed unpaired t-test). e, f Confocal images of P12 Yap1,Wwtr1iPCKO and littermate control lungs stained for PECAM1 (white). Note formation of capillaries at the secondary septa in controls (arrowheads) but not in mutants. Panels in f show higher magnification of insets in e. Scale bar, 20 µm (e) and 10 µm (f). g Three-dimensional high magnification reconstruction images of P12 Yap1,Wwtr1iPCKO and littermate control lungs stained for ERG (green), ICAM2 (red), and DAPI (blue). Scale bar, 30 µm. h Quantitation of ICAM2-stained vascular density based on 3D reconstruction surface images, as shown in g. Data represents mean ± s.e.m. (n = 4 mice; unpaired two tailed student t-test) i Maximum intensity projections of P7 Yap1,Wwtr1iPCKO and littermate control lungs stained for αSMA (red) and tropoelastin (blue). Scale bar, 100 µm. j Quantitation of staining intensity for αSMA or tropoelastin shown in i. Signal intensity was normalized to the size of the parenchymal region. Data represents mean ± s.e.m. (n = 4 mice; NS not significant, two-tailed unpaired t-test). k Western blot analysis of αSMA and tropoelastin in P7 Yap1,Wwtr1iPCKO and littermate control total lung lysates (n = 3 mice). Molecular weight marker (kDa) is indicated
