Anacardic acid inhibits TNF-dependent NF-κB activation. (A) Effect of anacardic acid per dose. KBM-5 cells were preincubated with indicated concentrations of anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA to test for NF-κB activation. (B) Effect of anacardic acid according to exposure duration. Cells were preincubated with 25 μmol/L anacardic acid for the indicated times, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA to test for NF-κB activation. (C) NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibody, preimmune serum, unlabeled NF-κB oligoprobe, or mutant oligoprobe and then assayed for NF-κB activation by EMSA. (D) The direct effect of anacardic acid on NF-κB complex was investigated. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nmol/L TNF and incubated for 30 minutes with the indicated concentrations of anacardic acid. They were then assayed for NF-κB activation by EMSA. (E) Anacardic acid blocks NF-κB activation induced by TNF, IL-1β, LPS, PMA, OA, and EGF. KBM-5 cells were preincubated with 25 μmol/L anacardic acid for 4 hours and then treated with 0.1 nmol/L TNF, 100 ng/mL IL-1β, or 10 μg/mL LPS for 30 minutes; 500 nmol/L OA for 4 hours or 25 μg/mL PMA or 100 ng/mL EGF for 2 hours. The cells were then analyzed for NF-κB activation by EMSA. (F-I) Inhibition of NF-κB activation by anacardic acid is not cell type–specific. H1299, Jurkat, Du145, and SCC4 cells were incubated with 25 μmol/L anacardic acid for 4 hours and then incubated with 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA.