Figure 3

CD46 induced autophagy in primary upper airway epithelial cells from the controls and asthmatic patients. Cells were incubated for 4 hours in complete medium in the presence of anti-CD46 mAb (5 μg/ml), isotype control antibody (IgG), or in nutrient-deprived media (starvation) and/or the autophagy inhibitor 3-methyladenine (3-MA) (10 mmol/L) (Sigma-Aldrich, St. Louis, MO). (A) Representative images of GFP-LC3 puncta (autophagosomes) in nasal epithelial cells are shown by confocal microscopy. (B) The cytosolic soluble form of LC3-I was converted into the autophagic vesicle-associated form of LC3-II and was used as a marker of autophagosome formation. The number of GFP-LC3 vesicles per cell in primary nasal epithelial cells was calculated from 200 cells for each experiment. Quantification of GFP-LC3 puncta per cell was assayed and the statistical data are shown. (C) Immunoblotting was used to analyze the LC3- II expression in primary upper airway epithelial cells from the controls and asthmatic patients. (D) Statistical data of the experiments with 30 paired samples as shown. (E) Primary nasal epithelial cells from the asthmatic subjects were treated with bafilomycin, and immunoblotting was used to analyze the expressions of LC3-II. (F) Statistical data of the experiments with six paired samples as shown. The Kruskal-Wallis test was used to determine significant differences. *p < 0.05.