Effect of Compound K on the requirement for caspase-8 for Compound K-induced apoptosis in HL-60 cells. (A) HL-60 cells were pretreated with/without 100 μM z-IETD-fmk (a specific caspase-8 inhibitor) for 1 h and then treated with 20 μM of Compound K for 4 h. Mitochondrial (M) and cytosolic (C) fractions were prepared as described in Methods. Caspase-3, Bid, and tBid were analyzed by Western blotting. Anti-α-tubulin, anti-COX IV and anti-β-actin were used as internal controls. (B) Samples were treated as described in the legend of Figure 5(A). Genomic DNA fragments were extracted and resolved on 2% agarose gel. Apoptotic DNA fragmentation was visualized by ethidium bromide staining. (C) Fas was immunoprecipitated from total protein lysates and proteins were subjected to Western blotting for anti-FasL, anti-FADD, or anti-caspase-8 antibodies (IP: immunoprecipitation, IB; immunoblotting).