TRAIL induces rapid production of DAG that is dependent on phospholipase activity. HeLa and Jurkat cells were either untreated (0 μmol/L; A and B) or preincubated with D609 (B) at various concentrations. Cells were then subjected to TRAIL (10 ng/mL) treatment for 3 hours and DAG content was measured as described in Materials and Methods. Columns, mean DAG levels of three independent experiments each done in duplicates; bars, SD. *, P < 0.05, statistically significant two-sided Wilcoxon test compared with control cells; **, 0.05 < P < 0.10, statistically significant two-sided Wilcoxon test compared with control untreated cells in HeLa and Jurkat cells.