Figure 1.
EGCs and GSNO attenuate Cytomix-induced monolayer permeability in Caco-2 cells.
Caco-2 cells were grown in either the presence or absence of EGCs or GSNO and incubated with Cytomix (TNF-α, IFN-γ, IL-1β) or PBS for 24 hours. L-NAME was used to block GSNO activity from EGCs. Caco-2 monolayer permeability to 4kDa FITC-Dextran was measured (n ≥ 4 samples per group). Cytomix-stimulation results in an increase in monolayer permeability, indicating barrier dysfunction. The presence of either EGCs or GSNO significantly reduces permeability levels. *p < 0.05 versus the controls Alone, + EGC, + GSNO ; **p < 0.01 versus + Cytomix, and +EGC +L-NAME +Cytomix.
Figure 2.
EGCs and GSNO improve localization of ZO-1 in Caco-2 and MDCK cells.
Epithelial cells were grown in either the presence or absence of EGCs or GSNO and incubated with Cytomix (TNF-α, IFN-γ, IL-1β) for PBS for 24 hours. A: Caco-2 monolayers stained with anti-ZO-1 antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have an altered localization of ZO-1 away from the cell surface compared to controls, indicating tight junction disruption. Stimulated cells co-cultured with EGCs or incubated with GSNO exhibited a restoration of ZO-1 localization at the cell surface. B: MDCK cells stained with anti-ZO-1 antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have an altered localization of ZO-1 indicating tight junction disruption. Stimulated cells co-cultured with EGCs or incubated with GSNO demonstrated normal distribution of ZO-1. Images are of 60x magnification and exposure matched. Bar = 30µm.
Figure 3.
EGCs and GSNO improve expression and localization of occludin in Caco-2 cells.
A–B: Caco-2 occludin immunoblot and relative band densities. Cytomix-stimulation decreased occludin expression compared to controls. Co-culture of stimulated-cells with EGCs or incubation with GSNO prevented the Cytomix-induced loss of occludin expression. C: Caco-2 monolayers stained with anti-occludin antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have an altered localization of occludin away from the cell surface compared to controls, indicating tight junction disruption. Stimulated cells co-cultured with EGCs or incubated with GSNO exhibited a restoration of occludin localization at the cell surface. Images are of 60x magnification and exposure matched. Bar = 30µm. *p < 0.05 versus Caco-2 cells alone, Caco-2 + EGC, Caco-2 + GSNO; **p < 0.05 versus Caco-2 + Cytomix using analysis of variance (ANOVA).
Figure 4.
EGCs and GSNO improve expression and localization of occludin in MDCK cells.
A–B: MDCK occludin immunoblot and relative band densities. Cytomix-stimulation decreased occludin expression compared to controls. Co-culture of stimulated-cells with EGCs or incubation with GSNO prevented the Cytomix-induced loss of occludin expression. C: Caco-2 monolayers stained with anti-occludin antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have an altered localization of occludin away from the cell surface compared to controls, indicating tight junction disruption. Stimulated cells co-cultured with EGCs or incubated with GSNO exhibited a restoration of occludin localization at the cell surface. Images are of 60x magnification and exposure matched. Bar = 30µm. *p < 0.05 versus MDCK cells alone, MDCK + EGC, MDCK + GSNO ; **p < 0.05 versus MDCK + Cytomix using analysis of variance (ANOVA).
Figure 5.
EGCs and GSNO reduce expression of P-MLC in Caco-2 cells.
A–B: Caco-2 P-MLC immunoblot and relative band densities. Cytomix-stimulation increased P-MLC expression compared to controls. Co-culture of stimulated-cells with EGCs or incubation with GSNO prevented the Cytomix-induced increase in P-MLC expression. C: Caco-2 monolayers stained with anti-P-MLC antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have increased levels of P-MLC compared to controls, indicating tight junction disruption. Co-culture of Cytomix-stimulated cells with EGCs or incubation with GSNO restores normal levels of P-MLC. Images are of 60x magnification and exposure matched. Bar = 30µm. *p < 0.05 versus Caco-2 cells alone, Caco-2 + EGC, Caco-2 + GSNO; **p < 0.05 versus Caco-2 + Cytomix using analysis of variance (ANOVA).
Figure 6.
EGCs and GSNO reduce expression of P-MLC in MDCK cells.
A–B: MDCK P-MLC immunoblot and relative band densities. Cytomix-stimulation increased P-MLC expression compared to controls. Co-culture of stimulated-cells with EGCs or incubation with GSNO prevented the Cytomix-induced increase in P-MLC expression. C: MDCK monolayers stained with anti-P-MLC antibodies (green) and DAPI (blue) and imaged through confocal microscopy. Cytomix-stimulated monolayers have increased levels of P-MLC compared to controls, indicating tight junction disruption. Co-culture of Cytomix-stimulated cells with EGCs or incubation with GSNO restores normal levels of P-MLC. Images are of 60x magnification and exposure matched. Bar = 30µm. *p < 0.05 versus MDCK cells alone, MDCK + EGC, MDCK + GSNO; **p < 0.05 versus MDCK + Cytomix using analysis of variance (ANOVA).