Figure 6

TRAIL plus chemotherapy activates the mitochondrial apoptosis pathway. (a) HepG2 cells were pretreated with or without JNK inhibitor (Inh) and the stimulated with medium control (Ctl) or doxorubicin (Dox; 1 μg/ml) plus TRAIL (10 ng/ml). Cells were lysed, Mcl-1 was immunoprecipitated and Bim bound to Mcl-1 was detected by western blot. Bim, Mcl-1 and tubulin levels in total cell lysates were analyzed for comparison. (b) Cells were treated with doxorubicin, TRAIL or both, and active Bax was immunoprecipitated and detected by western blot. Bax in total lysates was analyzed for comparison. (c) Cells were treated with doxorubicin, TRAIL or both, and cytoplasmic (C) and mitochondrial fractions (M) were isolated. Cytochrome c (Cyto c), SMAC and for comparison tubulin in the different fractions were analyzed by western blot. (d) Cells were transfected with control plasmid, Mcl-1, Bcl-xL or both, and then exposed to medium control or doxorubicin plus TRAIL. After 6 h DEVDase activity in cell lysates was analyzed. Mean values±S.D. of quadruplicates are shown. n=3. ^*P<0.005, ^**P<0.001, compared with control transfection