Figure 1

The transient inhibition of TNFα-induced NF-κB activation by H₂O₂. (A) HEK293 cells were transfected with an NF-κB reporter construct (IgκB-Luc) together with a control expression vector (β-actin/β-galactosidase) and incubated for 2 days. The cells were incubated with various doses of H₂O₂ in for 20 min in DPBS/D-glucose, washed and stimulated with TNFα (20 ng/ml) in DMEM/10% FBS for 4 h. The luciferase activity in the cell lysate was determined by luminometry and normalized to the β-gal activity (n = 3). (B) The cells were incubated with or without H₂O₂ (0.5 mM) for 20 min and then stimulated with TNFα for various times, and the luciferase activity was measured (n = 3). (C) The cells were treated as in (B), and cellular total RNA was isolated. cDNA was synthesized using MMLV reverse transcriptase, and quantitative real-time PCR was performed with primer and probe sets for IL-1β (upper panel) and ICAM-1 (lower panel) to calculate the mRNA level, which was then normalized to GAPDH (n = 3).