Fig. 1
SARS-CoV-2 envelope (E) protein disrupted barrier function and altered the expression of tight junction proteins in airway epithelial cells. a, b 16HBE14o- monolayers were stimulated with E protein (50 μg/ml) apically for the indicated time points. a Transepithelial resistance (TER) values were measured and shown relative to the TER before E protein stimulation (n = 5). b Fluorescence intensity of fluorescein isothiocyanate-dextran (4 kDa, FD4) flux across cell monolayers were monitored (n = 3). c Diagram of Pseudomonas aeruginosa strain PAO1 penetration through airway epithelial cell monolayer. Airway epithelial cells were seeded into the upper chamber of plate inserts with a 3 μm pore size membrane, which allowed the passage of PAO1. Cell monolayer with tight junctions (TJs) were infected apically with PAO1 and the penetrated bacteria from the basolateral medium in different groups were enumerated. The figure was created using Adobe Illustrator. d The colony-forming units (CFU) of penetrated PAO1 obtained from the plate counting technique. 16HBE14o- monolayers were infected with PAO1 at a multiplicity of infection (MOI) of 20 for 2 h, with or without E protein (50 μg/ml) stimulation (n = 3). e ICR mice were injected with FD4 after the stimulation of E protein (300 μg/ml) or saline for 6 h. The fluorescence ratio of BALF and serum was calculated (n = 6). f Heatmap of tight junction-related genes with significant changes in BEAS-2B cells stimulated with E protein (50 μg/ml) for 12 h. g Quantitative real-time PCR analyses showing the expression of ZO-1, occludin and claudin-4 in BEAS-2B cells stimulated with E protein (50 μg/ml) for the indicated time points (n = 3). Data are shown as means ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group. h Representative immunoblots showing the expression level of ZO-1, occludin and claudin-4 in BEAS-2B cells after stimulation with E protein (50 μg/ml) for the indicated time points. GAPDH served as a loading control. i Immunofluorescence images showing the expression of claudin-4 in BEAS-2B cells, in the absence or presence of E protein (50 μg/ml) stimulation. Scale bar = 20 μm. j Immunofluorescence staining of lung sections showing the expression of claudin-4 in Ad5-hACE2 transgenic mice with or without SARS-CoV-2 infection (1 × 105 PFU). Scale bar = 50 μm. k After incubation with E protein (50 μg/ml), the co-immunoprecipitates and the total lysates (Input) were analyzed by immunoblotting with the indicated antibodies
