Effects of TNF on the actin cytoskeleton and MLC phosphorylation.

<p>A) Analysis of the effects of TNF on actin/CL5 co-localization. Post-confluent HDMEC monolayers immunostained with anti-CL5 and phalloidin-stained for actin were imaged by fluorescence microscopy and analyzed for co-localization as described. Actin/CL5 co-localization was lost after TNF treatment for 8 hours at 0.8 ng/ml, resulting in a statistically significant difference in the Pearson correlation co-efficient (y-axis) by two-tailed t-test. B) Immunofluorescence microscopy representative of the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120075#pone.0120075.g004" target="_blank">Fig. 4</a>. Co-localization of the cortical actin cytoskeleton with junctional CL5 in DMSO control HDMEC (arrow in left panel; phalloidin staining, red; anti-CL5, green) is dissociated by TNF (center panel), a change prevented by Bay11 (right panel). Scale bar, 15 μm. C) Time course of TNF-induced changes in MLC (Thr18/Ser19) phosphorylation and ICAM-1 levels measured by immunoblotting with controls for total MLC and for β-actin. TNF treatment for the times indicated was at 10 ng/ml. D) Dose response of TNF-induced changes in phospho-MLC levels assessed by immunoblot analysis. HDMEC lysates were harvested at 6 hours of TNF. E) Effect of Bay11 on changes in P-MLC levels induced by TNF. Bay-11 was used at a 3 μM concentration. F) Effect of SR-IκB on changes in P-MLC levels induced by TNF. In E and F) TNF treatment was for 6 hours at 0.8 ng/ml. Note that Bay11 and SR-IκB each inhibit TNF-induced increases in ICAM-1 protein levels as well as MLC phosphorylation. Representative of 2 (B, E) or 3 (C, D, F) independent experiments with similar results.