(A) The right lungs from Dll4+/+ (n = 6) and Dll4+/lacZ (n = 4) mice were harvested on P6, and RNA extracted was used for RNA-Seq using an Illumina NovaSeq platform. Heatmap of 336 differentially expressed genes between Dll4+/+ (n = 4) and Dll4+/lacZ (n = 4) mouse lungs (P < 0.05). (B) qRT-PCR demonstrates Aqp5, Pdpn, Hopx,
Esr2, Klf2, Spc, and Pdgfrα expression in Dll4+/+ and Dll4+/lacZ mouse lungs at P4, P14, and P28. (C) AQP5 (green) and SPC (red) IF staining with DAPI (blue) in Dll4+/+ and Dll4+/lacZ mouse lungs at P14, with quantification shown for percentage of SPC+ cells of the total DAPI-stained cells (D), n = 6 in each group and P < 0.005. (E) Mouse lungs were harvested from Dll4+/+ and Dll4+/lacZ mice at P14 and P28. Lung homogenates were used to quantify SPC and PDGFRA by immunoblotting, with densitometry shown graphically (F), n = 5 mice per group; P < 0.01. (G) PDGFRA (brown) IHC staining with Harris staining (blue) on P8 and P14 Dll4+/+ and Dll4+/lacZ mouse lung sections, with quantification shown for percentage of PDGFRA+ cells in total Harris-stained cells (H), n = 6 in each group, *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar: 20 μm. Data are shown as mean ± SD. (B, F, and H) Gaussian distribution used 1-way ANOVA with Tukey’s test. (D) Gaussian distribution with 1 comparison used 2-tailed t test with Welch’s correction.