Fig. 4

Effect of constitutive KRAS activity on promoting 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 qPCR analysis to assess mRNA expression of KRAS to confirm over-expression of KRAS during ALI culture. Data points indicate the mean expression and error bars indicate standard error of the mean. Data from n = 3 independent experiments, each performed in triplicate with independent donors of BC. b Histology of Lenti control, WT KRAS and activated KRAS cells at ALI day 28. c Quantification of epithelial thickness of ALI day 28. Bars indicate epithelial thickness. Error bars indicate standard error. Data from n = 3 independent experiments, each performed with an independent donor of BC. d qPCR analysis to assess mRNA expression of the proliferation marker MKI67 at ALI day 28. Bars indicate the normalized mRNA expression. Error bars indicate standard error. Data from n = 3 independent experiments, each performed in triplicate with independent donors of BC. e qPCR analysis to assess mRNA expression of BC markers (KRT5, TP63), secretory cell markers (MUC5AC, MUC5B, SCGB1A1), ciliated cell markers (FOXJ1, DNAI1) and squamous cell markers (KRT6B, IVL) at ALI days 7, 14, and 28. Data from n = 3 independent experiments, each performed in triplicate with independent donors of BC. Bars indicate the normalized mRNA expression. Error bars indicate standard error of the mean. Asterisks indicate p < 0.05 (*) or p < 0.001 (**). ANOVA was used to determine the statistical significance among groups as described in the methods section