Fig 3. Effect of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-kB activity in human umbilical vascular endothelial cells (HUVECs).

(A) HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol for each indicated time point, followed by stimulation with 0.1 ng/ml TNF-α for an additional 15 min. IκB-α, NF-kB p65 and phospho-NF-kB p65 protein expression was analyzed via western blotting as described in the Materials and Methods. Representative blots from three independent experiments are shown. (B) 7-Ketocholesterol stimulates the nuclear translocation of the p65 subunit. Immunofluorescent images depict HUVECs stained with a mouse monoclonal antibody specific for p65 (green) and FITC-labeled goat anti-mouse secondary antibody. Nuclei were stained with 0.1 μg/ml DAPI (red) for 10 seconds before the cells were mounted on microscope slides. Representative merged photos from three independent experiments are shown.