Two week exposure to IL-4 alters the morphology and expression of markers for goblet cells and ciliated cells. (A) Expression of mucins and markers of ciliated cells at the mRNA level in ALI cultures exposed to various concentrations of IL-4 for two weeks. (B) QPCR analysis of MUC5AC in ALI cultures differentiated using IL-4 or IL-13. Data were normalized for expression against two reference genes (β-actin and GAPDH). (C) Upper panels: toluidin blue staining of an untreated (left upper panel) and IL-4-treated (10 ng/ml) ALI culture (right upper panel). Middle panels: transmission electron microscope picture of the same cultures. Lower panels: MUC5AC protein expression in an untreated and IL-4-treated ALI culture after staining with a mouse-anti-MUC5AC antibody (clone 45M1) and goat-anti-mouse IgG-Alexa488 as a secondary antibody. (D) MUC5AC dot blot of apical washes of ALI cultures stimulated with 10 or 20 ng/ml of IL-4 of one donor. Increase in MUC5AC protein was observed in five out of six experiments using cells from four different donors.