Figure 2.

Corticotropin releasing factor receptor 2 signaling increases trans-epithelial permeability in early-differentiated HT-29 cells. A: The trans-cellular flux of dextran-FITC (4 kDa) was measured by spectrofluorimetry. HT-29 cells were cultured during 10 d in Gal medium. After 5 h treatment or not (untreated cells) with 100 nmol/L Ucn3, dextran-FITC was added at the apical compartment. Then the green fluorescence incorporated in the cell monolayer was measured after 10, 20, and 30 min. The release of fluorescence in the basal compartment is associated to trans-cellular permeability. Ucn3 treatment seems to increase transcellular transport in early-differentiated HT-29 cells. Two-way ANOVA, P < 0.01; B: Phase contrast analysis of early differentiated HT-29 cells exposed or not to Ucn3 (100 nmol/L). (Scale bar: 25 μm). Red arrows indicate the formation of refractile structures inside the epithelial monolayer; C: Confocal imaging from basal to apical poles of phaloidin-TRITC labeling (upper panel) and Differential Interference Contrast (lower panel) of early-differentiated HT-29 cells treated 2 h with 100 nmol/L Ucn3 (Scale bar: 10 μm). White arrows indicate refractile structures as observed before by phase contrast microscopy, while yellow arrows show alterations in cell-cell contacts. Acquisitions were performed on a Leica TCS SPE confocal microscope (objective × 100).