Fig. 6: The epithelial transcytosis of EcN OMVs involves pinocytosis and dynamin-dependent pathways.

a The mechanism underlying the ability of EcN OMVs to penetrate the gut epithelial cell barrier was studied using an in vitro intestinal epithelium model of Caco-2 monolayers grown in Transwell cultures. Various inhibitors targeting different endocytosis pathways and RFP-loaded OMVs were sequentially added to the upper chamber. Created in BioRender. Gong, X. (2025) https://BioRender.com/d95j675. b To determine whether epithelial transcytosis of OMVs occurs through an active or passive mechanism, RFP-loaded OMVs were incubated in the apical compartment of Transwell chambers at 37 °C or 4 °C for 4 hours. Fluorescence signals were detected in the basolateral chamber, and further normalized to that of the 37 °C group (n = 3 biologically independent samples). c The mechanism underlying epithelial transcytosis of OMV was examined by focusing on the first endocytosis step of transcytosis, using various chemical inhibitors targeting different endocytosis pathways at various concentrations (low, medium and high concentrations), including the vehicle (dimethyl sulfoxide, DMSO), chlorpromazine (inhibition of clathrin-dependent endocytosis), cytochalasin D (inhibition of membrane fusion), wortmannin (inhibition of phagocytosis), amiloride (inhibition of pinocytosis), nystatin (inhibition of caveolin-mediated endocytosis and lipid raft formation), and dynasore (inhibitors of dynamin-dependent endocytosis). Low concentration: 0.05 μM wortmannin; 0.25 μM cytochalasin D; 10 μM chlorpromazine; 20 μM amiloride; 0.1 μM nystatin and 20 μM dynasore; Medium concentration: 0.1 μM wortmannin; 0.5 μM cytochalasin D; 20 μM chlorpromazine; 100 μM amiloride; 0.2 μM nystatin and 100 μM dynasore; High concentration: 1 μM wortmannin; 2 μM cytochalasin D; 50 μM chlorpromazine; 200 μM amiloride; 1 μM nystatin and 200 μM dynasore. Fluorescence signals were detected in the basolateral chamber and were further normalized to that of the DMSO group (n = 3 biologically independent samples). d The permeability of the epithelial monolayers after incubation with OMVs along with DMSO or inhibitors was assayed with Lucifer Yellow (n = 3 biologically independent samples). The data are presented as the mean ± SD. The P value was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.