Fig. 5

Consequences of constitutive KRAS activity on promotion of basal cell (BC) differentiation into secretory and ciliated cells Primary human airway BC were infected with control lentivirus or lentivirus over-expressing wild-type (WT) KRAS or the constitutively active G12 V mutant (activated) and cultured on ALI for 28 days to assess the impact of KRAS on BC differentiation into a mucociliated epithelium. a Immunofluorescence staining of KRT5⁺ BC. Sections of cells on ALI day 28 membranes were stained for KRT5 (red) and DAPI (nuclei, blue). b Alcian blue staining of secretory cells. Sections of cells on ALI day 28 membranes were stained for Alcian blue (blue). c Immunofluorescence staining of MUC5B⁺ secretory cells. Sections of cells on ALI day 28 membranes were stained for MUC5B (red) and DAPI (nuclei, blue). d Immunofluorescence staining of SCGB1A1⁺ secretory cells. Sections of cells on ALI day 28 membranes were stained for SCGB1A1 (red) and DAPI (nuclei, blue). e Immunofluorescence staining of β-tubulin IV⁺ ciliated cells. Sections of cells on ALI day 28 membranes were stained for β-tubulin IV (green) and DAPI (nuclei, blue). f Immunohistochemical staining of IVL⁺ squamous cells. Sections of cells on ALI day 28 membranes were stained for IVL (red) and DAPI (nuclei, blue). The data for a-f are the mean for n = 3 independent experiments performed with independent donors of BC. The bars indicate the mean percentage of positively stained cells for n = 3 independent experiments, and error bars indicate standard error of the mean. ANOVA was used for statistical comparisons among groups as described in the Methods. Scale bar 20 μm