GA potentiates apoptotic effects of TNF and chemotherapeutic agents. (A) The chemical structure of GA. (B-D) GA enhances TNF-, 5-FU–, and doxorubicin-induced cytotoxicity. Five thousand cells were seeded in triplicate in 96-well plates. The cells were pretreated with the indicated concentrations of GA and then incubated with chemotherapeutic agents. Cell viability was then analyzed by the MTT method. (E) GA enhances TNF-induced cytotoxicity. KBM-5 cells were pretreated with indicated concentrations of GA for 4 hours and then incubated with 1 nM TNF for 24 hours. The cells were stained with a live/dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope (left panel). The percentage of apoptosis was plotted as mean plus or minus SD (right panel). (F) GA enhances TNF-induced annexin V–FITC binding. (G) Effect of GA on PARP cleavage. Cells were pretreated with 1.0 μM GA for 4 hours and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting with an anti-PARP antibody. (H) A293 cells were transiently transfected with p65 plasmids. After 24 hours, cells were treated with 1.0 μM GA for 4 hours followed by 1 nM TNF for 24 hours. Whole-cell extracts were prepared and analyzed by Western blotting with an anti-PARP antibody.