Effect of I3C on TNF-induced NF-κB activation. (A) I3C blocks NF-κB activation induced by TNF, PMA, LPS, and CSC. Jurkat cells were preincubated with 50 μM I3C for 24 hours; treated with 0.1 nM TNF and 10 μg/mL LPS for 30 minutes; treated with 100 ng/mL IL-1β, 15 ng/mL PMA, and 1 μg/mL CSC for 1 hour; and then analyzed for NF-κB activation as described in “Materials and methods.” (B) Jurkat cells were preincubated at 37°C with 50 μM I3C for the indicated times and then treated with 0.1 nM TNF at 37°C for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. Cell viability was determined by the trypan blue dye exclusion method. (C) I3C suppresses TNF-induced NF-κB in a dose-dependent manner in Jurkat, KBM-5, and H1299 cells. Cells were incubated with different concentrations of I3C for 24 hours, followed by an incubation with 0.1 nM TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. (D) I3C inhibits constitutive NF-κB activation. MM.1, U266, and SCC-4 cells were incubated with different concentrations of I3C for 24 hours. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA.