Molecular mechanisms of AZ-TAK1 antiproliferative activity lymphoma. (A) MCL cell lines were incubated with medium or 0.5μM of AZ-TAK1 for 24-48 hours. Whole-cell lysates were examined by Western blot for changes in intracellular proteins. AZ-TAK1 decreased TAK1 and p38 phosphorylation, indicative of their inactivation. AZ-TAK1 inactivated NF-κB, as indicated by the decrease in nuclear p65 and inhibition of IκB phosphorylation. (B) AZ-TAK1 down-regulated XIAP and activated caspase 9 and caspase 3. (C) AZ-TAK1–induced cell death was either partially or completely blocked by the caspase 9 inhibitor Z-LEHD-FMK. Each value represents a mean of 3 independent experiments done in triplicate (± SEM). (D) AZ-TAK1 released cytochrome c and SMAC/Diablo from the mitochondria in MCL cell lines (top). Western blot analysis was performed on subcellular mitochondrial fractions after incubation with AZ-TAK1 (0.5μM) for 48 hours. Cytofluorimetric analysis of MCL cell lines mitochondrial potential, as assessed with the mitochondrial specific cationic dye (middle), as described in “Evaluation of mitochondrial membrane potential.” In this representative experiment, treatment with AZ-TAK resulted in mitochondria depolarization in 75% of the cells, compared with control 8% in DMSO-treated cells. Summary of 3 independent experiments demonstrating the effect of AZ-TAK1 on mitochondria membrane polarization (bottom). Each value represents the percentage of cells with depolarized mitochondria. Cells were incubated for 12 hours in the absence (DMSO) or presence of AZ-TAK1 0.5μM.