Fig 4. Effects of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced mitogen-activated protein kinase (MAPK) activity in human umbilical vascular endothelial cells (HUVECs).

(A) HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol for each indicated time interval, followed by stimulation with 0.1 ng/ml TNF-α for an additional 15 min. Western blotting was used to evaluate the levels of p38, phospho-p38, JNK and phospho-JNK proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, followed by incubation with 5 μM p38MAPK inhibitor (SB203580) for 30 min and stimulation with TNF-α for an additional 4 h. A non-static adhesion assay was performed. Fluorescently labeled THP-1 cells were added to the HUVECs and allowed to adhere for 10 min under rotating conditions. Data are shown as means ± standard errors of the means (SEM). *p < 0.05 by a one-way analysis of variance followed by Tukey’s test. (C) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, incubated with 5 μM p38 MAPK inhibitor (SB203580) for 30 min and stimulated with TNF-α for an additional 4 h. Western blotting was used to evaluate the expression of E-selectin, ICAM-1, and VCAM-1 proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown.