Zalypsis induces changes in the cell cycle profile and apoptosis in MM1S cells. (A) MM1S cells were incubated with Zalypsis (5 nM) for 6, 12, and 24 hours, and the cell cycle profile was examined by flow cytometry after propidium iodide staining. Bars represent the percentage of cells in each phase of the cell cycle from a representative example. (B) MM1S cells were treated with Zalypsis (5 nM) for different time points, and the induction of apoptosis was analyzed by flow cytometry after staining with annexin V. (C) MM1S cells were treated with Zalypsis (5 nM) for the indicated times. DNA of the treated cells was isolated, and its fragmentation was analyzed by agarose gel electrophoresis. The position of the molecular weight markers is shown at the right. (D) MM1S cells were treated with Zalypsis (5 nM) for 12 and 24 hours, and the disruption of the mitochondrial membrane potential (ΔΨ_m) was analyzed by flow cytometry after staining with [DiOC₆(3)^low]. (E) MM1S cells were treated for the indicated times with Zalypsis, and cytochrome C, AIF, and endonuclease G in the mitochondrial fraction were analyzed by Western blotting. (F) The status of some Bcl-2 family members was analyzed by Western blotting after treatment of MM1S cells with 5 nM Zalypsis for the indicated time points in the presence or absence of the caspase inhibitor Z-VAD-FMK (50 μM for 60 minutes). (G) MM1S cells were treated with Zalypsis (5 nM) for the indicated times, and expression of PARP, caspase-3, caspase-7, caspase-8, and caspase-9 proteins was analyzed by Western blotting. (H) MM1S cells were plated and pretreated with the different caspase inhibitors (50 μM) for 60 minutes. Zalypsis (5 nM) was added to the corresponding samples, and the experiment was continued for 12 and 24 hours. Induction of apoptosis was analyzed by means of annexin V–FITC staining.