Fig 6. Activation and role of c-JNK in MK591-induced apoptosis.

C4-2B cells were plated as in Fig 5 and treated with MK591 (30 μM) for varying periods of time and changes in the phosphorylation of c-JNK was detected by Western blot (a). Then, the membrane was stripped and re-probed with regular c-JNK antibody to detect total c-JNK protein. Beta-actin was used as loading control. In (b), to test the role of c-JNK in apoptosis, the cells were pre-treated with SP600125 (a c-JNK inhibitor) or U0126 (a MEKK inhibitor) for 30 minutes. Then, the cells were treated with MK591 and apoptosis was measured by sandwich ELISA. Results are shown as mean values of each data point ± standard error (**p < 0.005, n = 4). In (c), effect of MK591 on mitochondrial integrity was examined by treating cells with MK591 for 16 hours followed by mitotracker-red for 15 minutes in the dark. DAPI (blue) was used to stain the nuclei. Representative pictures are shown here from three independent experiments with similar results. Note: A dramatic loss of mitochondrial membrane-potential was observed with MK591 treatment while ibuprofen was found ineffective.