FIG. 7.

TNF-α increases cell permeability in a PKCζ-dependent manner. BRECs were grown to confluence on transwell filters and then treated with (A) 2 μmol/l LY294002 (LY), (B) 10 μmol/l PKCζI-1, and (C) 250 nmol/l PKCζ pseudosubstrate inhibitor (PKCζp). The monolayer permeability to 70 kDa dextran was measured as described. Results represent the mean ± SEM of at least five experiments and are expressed relative to Ctrl. D–F: PKCζI-1 prevents tight junction complex disruption induced by TNF-α. Whole-cell extracts were assayed for (D) ZO-1, (E) claudin-5, and (F) occludin immunoreactivity by Western blotting. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least eight independent experiments and are expressed as the relative amount compared with control (Ctrl). All inhibitors were added to the cell culture medium 30 min before TNF-α addition (5 ng/ml, 6 h). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from control; #P < 0.05, ##P < 0.01, ###P < 0.001, significantly different form TNF-α, as determined by ANOVA followed by Bonferroni post hoc test.