Fig. 3.

Nanoparticles compound respiratory syncytial virus (RSV)-induced apical junctional complex (AJC) disruption in vitro. Epithelial cells were exposed to RSV (MOI = 0.5) and/or titanium dioxide nanoparticles (TiO₂-NP; 50 μg/mL). AJC proteins zonula occludens-1 (ZO-1), occludin, β-catenin, and E-cadherin were immunofluorescently labeled (A). Arrows indicate disrupted AJC induced by RSV or TiO₂-NP; arrowheads indicate potentiated disruption. Permeability of the barrier was examined by transepithelial electrical resistance (TEER) at 48 h, and changes were normalized to the time zero for each group (B) and FITC-dextran flux assay, for which changes were normalized to the untreated control group (C). 16HBE or human primary epithelial cells were apically exposed to TiO₂-NP for 48 or 96 h, respectively. Lactate dehydrogenase (LDH) assay and Western blot analysis of cleaved caspase 3 in 16HBE (D and F) and in human primary epithelial cells (E–G) were performed using supernatants and cell lysates, respectively. 16HBE (H) and human primary epithelial cells (I) were exposed to TiO₂-NP and/or RSV. Cell lysates were collected and expression of AJC proteins ZO-1, occludin, β-catenin, and E-cadherin was analyzed by Western blotting; n = 2. Densitometry using ImageJ revealed no significant differences among the experimental groups (data not shown). Scale bar = 20 μm. Data are presented as means ± SE; n = 3, *P < 0.05, ***P < 0.001 vs. untreated control as determined.