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. 2005 Feb;166(2):409–419. doi: 10.1016/s0002-9440(10)62264-x

Figure 5.

Figure 5

Low dose SSA prevents morphological tight junction disruption, increased MLC phosphorylation, and increased MLCK expression. A: Caco-2 monolayers were treated with IFN-γ (10 ng/ml) for 24 hours followed by TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA for 8 hours. Tight junction proteins (ZO-1, occludin, and claudin-1) were detected by immunofluorescence microscopy. 0.5 mmol/L SSA completely prevented ZO-1, occludin, and claudin-1 redistribution in IFN-γ-primed TNF-α-treated monolayers. Data are representative of three similar experiments, each performed in triplicate. B: Caco-2 monolayers were incubated with IFN-γ (10 ng/ml) for 24 hours and then transferred to media with TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA, 2 mmol/L SSA, or 30 μmol/L MG132, as indicated. Monolayers were harvested 8 hours after TNF-α addition. Lysates were assayed for phosphorylated MLC by SDS-PAGE immunoblot. SSA (0.5 mmol/L) prevented increases in MLC phosphorylation induced by IFN-γ and TNF-α (P < 0.01). In contrast, 2 mmol/L SSA and 30 μmol/L MG132 failed to prevent increases in MLC phosphorylation (P > 0.05 for 2 mmol/L SSA, P = 0.04 for 30 μmol/L MG132) (n = 2 in this representative experiment). C: Caco-2 monolayers were incubated with IFN-γ (10 ng/ml) for 24 hours and then transferred to media with TNF-α (2.5 ng/ml) and 0.5 mmol/L SSA, 2 mmol/L SSA, or 30 μmol/L MG132, as indicated. Monolayers were harvested 8 hours after TNF-α addition. Lysates were assayed for MLCK by SDS-PAGE immunoblot. SSA (0.5 mmol/L) prevented increases in MLCK expression induced by IFN-γ and TNF-α (P < 0.02). In contrast, 2 mmol/L SSA and 30 μmol/L MG132 failed to prevent increases in MLCK expression (P > 0.05 for 2 mmol/L SSA or 30 μmol/L MG132) (n = 2 in this representative experiment).