NF-κB Activation in VSMCs

To examine the nuclear factor (NF)-κB activation, the VSMCs were transfected with a reporter plasmid containing the luciferase reporter gene linked to five repeats of the NF-κB binding sites, as previously described [15 (link)]. Briefly, the VSMCs (1 × 10⁵ cells/well) were cultured to approximately 70% confluence in 24-well plates. Cells were then transiently co-transfected with 1 µg of NF-κB-luciferase reporter plasmid and 1 µg of β-galactosidase plasmid using Lipofectamine plus (Invitrogen, Carlsbad, CA, USA). At 6 hours after transfection, cells were starved for 48 hours, and then exposed to 15 µM lysoPC for the indicated time periods. Luciferase activity was measured with a luciferase assay kit (Promega, Madison, WI, USA) with signal detection for 5 seconds in a luminometer (Panomics Inc., Fremont, CA, USA). A β-galactosidase enzyme assay (Promega) was applied to determine the β-galactosidase activity at 420 nm with a SmartSpec 3000 spectrophotometer (Bio-Rad). The results are expressed relative to the NF-κB activity compared with controls after normalizing for β-galactosidase activity and protein concentration.