FIGURE 6.

Caspase 8 is required for TRAIL-mediated apoptosis in MCF7 cells. A, MCF7 cells were treated with 100 ng/ml TRAIL for 24 h, and protein lysates were prepared as described under “Experimental Procedures.” A total of 30 μg of protein was loaded, and Western blotting was performed using anti-caspase 8 and PARP antibody. B, to block the activation of caspase cascades, MCF7-Smad7 cells were pretreated with 10 μm of the pan-caspase inhibitor (Z-VAD-fmk) or the caspase 8-specific inhibitor (Z-IETD-fmk) for 2 h. Cells were then treated with TRAIL for 24 h, and activation of caspase was measured by analyzing degradation of PARP and caspase 8. C, after treatment with the caspase inhibitor and TRAIL, DNA fragmentation was measured by preparing the genomic DNA. The DNA was run onto 2% agarose gel and visualized by staining with EtBr. D, after knockdown of caspase 8 by lentiviral shRNA, MCF7-Smad7 cells were treated with TRAIL for 24 h. PARP and caspase 8 were detected with specific endogenous antibody, and β-actin was used as normalization control.