Transient transfection and luciferase assays

Cells (1 × 10⁵) were plated in 6-well plates overnight prior to transfection. Then, the cells were co-transfected with 2 μg of the NF-κB-luc reporter construct and 0.1 μg of the renilla luciferase-expression plasmid using 6 μl of FuGENE 6. At 48-hours post-transfection, the cells were treated with sB7-H3, TAK-242 or BAY11-7028 and then lysed, and the activities of firefly and renilla luciferases were assessed using a dual-luciferase reporter assay system. All the experiments were performed in triplicate.