Additional file 1: of Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD

Figure S1. Differentiated primary human bAEC cultures closely approximate the airway epithelium in vivo. An important requisite for assessing the epithelial barrier is establishing a model that closely approximates the epithelium in the human airways. A. Transmission electron microscopy of the apical margin of a bronchial airway epithelial cell (bAEC) grown at an air-liquid interface (ALI) exhibits the features of ciliated bAEC found in the human airway, with columnar morphology, cilia (C) containing microtubules (MT; a diagnostic feature of cilium), and the smaller villi (V) projecting from the apical membrane. Note that the mucus blanket produced by goblet cells is also present but is lost during sample preparation. B. Scanning electron microscopy of a fully differentiated epithelial culture showing cilia and the smaller plasma membrane villi projection. In this example, the epithelial layer supports primary human T cells that were co-cultured on the epithelium for 24 h. Not shown is high speed live cell imaging of cilia beating at a frequency of 10 Hz, and the directional movement of activated charcoal suspended in media on the epithelial layer, which is indicative of synchronous cell-to-cell co-ordination via communication through gap junction complexes. C. Confocal immunofluorescence analysis of a primary human differentiated bAEC culture shows an extensive network of apicolateral tight junction complexes that maintain the selectively permeable epithelial barrier. Here staining is shown for the essential tight junction protein ZO-1. Underlying nuclei are resolved (albeit out of focus to favour the resolution of the ZO-1 fluorescent signal) using the nucleic acid stain DAPI (4′,6-diamidino-2-phenylindole). D. A SEM micrograph of AEC grown at an ALI before the production of cilia (six days post air exposure), showing the defined apicolateral margins between adjacent cells formed by tight junction complexes (three shown with white arrows). In this example the epithelial layer supports primary human alveolar macrophages (Alv Mac) that were co-cultured on the epithelium for 24 h. (TIF 7742 kb