Compound K caused a loss in mitochondrial membrane potential and induced the translocations of cytochrome c and Smac/DIABLO from mitochondria to the cytoplasm in HL-60 cells. (A) Cells were pretreated with 5 μM cyclosporin A (CsA) for 30 min and then treated with 20 μM of Compound K for 2 h, stained with DiOC6, and analyzed by flow cytometry. CCCP (100 μM) was used as a positive control. (B) Cells were pretreated as described in the legend of Figure 3 (A). Cleavages of procaspase-8 and -3 were analyzed by Western blotting. DNA fragmentation was measured using (C) DAPI assays and (D) fragmented genomic DNA was extracted and resolved on 2% agarose gel. Data presented are the means ± SD of three independent experiments. ***P < 0.001 vs. untreated controls, †
P < 0.05 vs. Compound K-treated cells; significances were determined using the Student's t-test. (E) Cells were harvested after being incubated with Compound K at 20 μM for the indicated times. Mitochondrial (M) and cytosolic (C) fractions were prepared as described in Methods. Cytochrome c, Smac/DIABLO, and XIAP were analyzed by Western blotting. α-tubulin, cytochrome c oxidase (COX) IV, and β-actin were used as internal controls. (F) Compound K induced an interaction between XIAP and Smac/DIABLO. Cells were treated with 20 μM of Compound K for the indicated times. Smac/DIABLO was immunoprecipitated from total protein lysates and proteins were subjected to Western blotting for anti-XIAP antibody (IP: immunoprecipitation, IB; immunoblotting).