Figure 1. Stichoposide D (STD) induces apoptosis of K562 and HL-60 cells through the activation of ceramide synthase 6 (CerS6).

A. Structure of STD. B. K562 and HL-60 cells (1 × 10⁵ cells/well) were each incubated with various concentrations (0, 0.3, 0.5, 1.0, 1.5 μM) of STD for 24 h or 6 h. After treatment for 24 h, cell viability was determined by MTT assay (upper panel). After treatment for 6 h, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining (lower panel). These data represent the mean ± SD of three independent experiments. IC₅₀ of STD in each cell is indicated. *P < 0.05, **P < 0.01, ^***P < 0.001 versus control. C–F. K562 and HL-60 cells were transiently transfected by electroporation with no siRNA (shock), nonspecific control (NC) siRNA, CerS6 siRNA-1, or CerS6 siRNA-2 for 48 h. (C) Western blot analysis of protein lysates. (D) Transfected K562 cells were exposed to 1.0 μM STD for 2 h and fixed. After permeabilization, samples were stained with PE-anti-CerS6, ceramide, or Fas antibodies and with Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (E) Left panel: The culture medium was changed, and K562 and HL-60 cells were treated with or without STD (1.0 μM and 1.5 μM, respectively) for 6 h. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Results are the mean ± SD of three independent experiments. *P < 0.05, ^***P < 0.001, cells treated with STD alone versus cells transfected with CerS6–1 or CerS6–2 siRNA and treated with STD. Right panel: Cells stained with DiOC₆. Reduction of Djm was determined by monitoring DiOC₆ uptake using flow cytometry. Low Djm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. Results are the mean ± SD of three independent experiments. ^***P < 0.001 for cells treated with STD alone vs. cells transfected with CerS6–1 siRNA and treated with STD. (F) Western blot for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase (COX IV) was used as a mitochondrial marker. Western blots (C, F) 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.