NF-κB reporter assay

The NF-κB reporter assay was described previously [19]. The NF-κB reporter plasmid pGL4.32 containing an NF-κB response element that drives the transcription of the luciferase reporter gene luc2P, the luciferase control plasmid pGL4.74 (Promega, Madison, WI), and the pEGFP-effector plasmid were co-transfected into HeLa cells using Fugene HD (Promega), according to the manufacturer’s protocol. Forty-eight hrs later, the cells were stimulated with 10 ng/ml TNF-α (Peprotech, Rocky Hill, NJ) for 30 min. NF-κB activity was calculated using a Dual-Glo Assay System (Promega) according to the manufacturer’s instructions.

For the NF-κB reporter assay in HeLa cells infected with S. Typhimurium, pGL4.32 and pGL4.74 were transfected into HeLa cells which were seeded at 2.5 × 10⁴ cells/well in 24-well plates for 24 hrs. Forty-eight hrs after the transfection, the cells were infected with S. Typhimurium, which was incubated in LB containing 0.3M NaCl to induce T3SS-1 [26], and the gentamycin killing assay was performed as described previously [19]. After 4 or 20 hrs of infection, the cells were washed three times with PBS and lysed with 1 × Passive Lysis Buffer (Promega). NF-κB activity was calculated as described above.