Figure 5. Clustering of Fas and its downstream molecules in lipid rafts during STD-induced apoptosis.

A. K562 and HL-60 cells were pretreated with MβCD (20 μg/ml) and nystatin (20 μg/ml) for 1 h and cultured in medium containing STD (1.0 μM, 1.5 μM, respectively) for 6 h. After treatment for the indicated times, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining. Upper panel: representative of three independent experiments. Lower panel: mean ± SD of three independent experiments. **P < 0.01; ^***P < 0.001 versus STD-treated cells. B. Whole cell lysates from K562 cells incubated with STD for 6 h in the presence or absence of MβCD were prepared in parallel and subjected to Western blot analysis. C. K562 cells were treated with STD in the presence or absence of MβCD or nystatin for 2 h and fixed. After permeabilization, samples were stained with PE-anti-Fas, CerS6, ceramide, p-p38, or cleaved caspase-8 antibodies and Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. D. Cells were pretreated with MβCD or nystatin for 1 h before treatment with STD for 6 h. Cells were stained with DiOC₆, and reduction of Δψm was determined by monitoring DiOC₆ uptake using flow cytometry. Low Δψm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC₆ assays represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus STD-treated cells. E. Western blot analysis for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Western blots (B, E) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.