Fig. 4

Spatio-temporal changes of epithelial cell monolayer following treatment with NE. Confluent monolayers of 16HBE epithelial cells were incubated alone or in the presence of defined NE concentration (200 nM). Briefly, cells were first subjected to immunofluorescence staining of E-cad as described in material and methods. Next, cell monolayers were left untreated or exposed to NE (200 nM) alone or pre-incubated with SLPI (400 nM). Video recording was performed for 6 h. a-b, Representative phase contrast images of untreated (a) or NE-exposed (b) 16HBE cells. Unlike control cells with flat polygonal morphology, NE-treated cells detached from each other and displayed rounded morphology. c-d, Representative immunofluorescence images of untreated or NE-exposed 16HBE cells. Immunofluorescent staining showed E-cad surrounding uniformly cohesive cells (c). In contrast, cells exposed to NE lost their immunostaining for E-cad coinciding with the appearance of intercellular gaps (d). Of note, NE preincubated with SLPI (e) or SLPI alone (f) had less striking effect on E-cad distribution and monolayer integrity. Asterisks depict formed gaps. Scale bar = 40 μm. Experiments were repeated twice with similar observations