Down-regulation of p300 HAT abrogates the effect of anacardic acid. (A) TNF regulates antiapoptotic gene expression. The wild-type and p65^−/− MEF cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using the relevant antibodies. (B) Anacardic acid inhibits binding of NF-κB to the COX-2 and MMP-9 promoter. KBM-5 cells were pretreated with 25 μmol/L anacardic acid for 4 hours and treated with 1 nmol/L TNF for the indicated times, and the proteins were cross-linked with DNA by formaldehyde and then subjected to chromatin immunoprecipitation (ChIP) assay using an anti-p65 antibody with the COX-2 and MMP-9 primers. Reaction products were resolved by electrophoresis. (C) Down-regulation of p300 by RNA interference reverses the effect of anacardic acid. A293 cells were transfected with indicated concentration of p300 siRNA or scrambled (SC) control. After 48 hours, cells were harvested, and whole-cell extracts were prepared and analyzed by Western blotting with an anti-p300 antibody. (D) Transfected cells were preincubated with 25 μmol/L anacardic acid for 4 hours and then treated with 0.1 nmol/L TNF for 30 minutes. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA.