Anacardic acid potentiates apoptosis induced by TNF and chemotherapeutic agents. (A) Structure of anacardic acid (AA). (B) Anacardic acid potentiates TNF-induced apoptosis. KBM-5 cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. The cells were stained with the Live/Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope. The results shown are representative of 3 independent experiments. (C) Cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. Cells were incubated with an anti–annexin V antibody conjugated with FITC and then analyzed by flow cytometry for early apoptotic effects. (D) Cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometry for apoptotic effects. (E) KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 24 hours. Whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. (F) 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 an anti-PARP antibody.