FIG 4.

GSNO contributes to the maintenance of epithelial tightness of rotavirus-infected polarized Caco-2 cells and induces increased expression of ZO-1. (A) Polarized Caco-2 cells (>450 Ω/cm²) were stimulated with GSNO for 24 h and infected with rotavirus. At 6 h p.i., the apical medium was replaced with medium containing 4-kDa FITC-dextran (2 mg/Transwell insert), and samples were obtained from the basolateral side at 22 h p.i. to measure passage of FITC-dextran. The controls were noninfected cells with and without GSNO and infected cells without GSNO. Basolateral samples were analyzed for FITC-dextran by spectrophotometry (494/518 nm), and values are presented as the optical density (OD) values and means with SD. *, P < 0.05, unpaired t test (n = 5 or 6). (B) GSNO increases ZO-1 tight junction protein in Caco-2 cell monolayers. Caco-2 cell monolayers cultured on Lab-Tek II chamber slides to near confluence with and without 24-h exposure to GSNO were stained for ZO-1 expression (red; Alexa Fluor 594). Confocal images were captured and the mean intensities on single-cell circumference were measured using ImageJ. (C and D) Monolayers of GSNO-treated (C) and untreated (D) Caco-2 cells. Data are mean fluorescence intensities and means with SD. ***, P < 0.001, Mann-Whitney test (n = 17). (E and F) Deconvolved images visualized in ortho-mode with XZ and YZ intensities of GSNO-treated (E) and untreated (F) Caco-2 cells. The XZ and YZ selections in each image are positioned to cross the maximum number of high-intensity ZO-1-labeled cell borders.