Fig. 3.

TNF-α stimulation of MCP-1 expression involves cross talk between the ERK and NF-κB pathways. A: U0126 downregulates TNF-α-stimulated NF-κB transcription. MCs were cotransfected with a chimeric NF-κB promoter-luciferase construct (NF-κB/pGL2) and a Renilla luciferase control plasmid (phRG-Basic) for 8 h and treated with U0126 (25 μM) for 1 h followed by TNF-α (10 ng/ml) for 6 h. Luciferase activity was assessed using the Dual Luciferase Reporter Assay System (Promega). Values are expressed as means ± SE relative to TNF-α-treated cells; n = 3 experiments. #P < 0.05 vs. control. *P < 0.05 vs. TNF-α-treated cells. B: U0126 decreases TNF-α-stimulated nuclear translocation of NF-κB. MCs were pretreated with U0126 for 30 min and then stimulated with TNF-α for 1 h. Cells were fixed and immunostained for the p65 subunit of NF-κB, and nuclei were stained with DAPI. Slides were analyzed by confocal microscopy. 1: Vehicle control. 2: Vehicle + TNF-α. 3: U0126 + TNF-α. Insets: same images without DAPI stain overlay.