MLN8237 induces accumulation of G₂/M phase and apoptosis in MM cells. (A) DNA profiles of MM cell lines, MM1.S and OPM1, as well as tumor cells from MM patient BM treated with DMSO or MLN8237 (0.5μM) for 24 hours were evaluated by flow cytometry. The percentage of cells in G₀G₁, G₂M, and S phases are shown. (B) MM cell lines, RPMI8226 and OPM1, as well as tumor cells from MM patient BM were cultured with DMSO or 0.5 to 1μM of MLN8237 for 24, 48, and 72 hours. Induction of apoptosis and cell death were determined by flow cytometric analysis in MM cells costained with annexin V and PI. (C) Induction of PARP, caspase-9, and caspase-3 cleavage by MLN8237 was measured by immunoblotting in MM cell lines. (D) Representative photomicrograph of β-galactosidase activation in MLN8237 (0.5μM, 48 hours) treated MM1.S cells. Arrows indicate activated β-galactosidase (blue) in cytoplasm. The senescent MM1.S cells (β-galactosidase active, blue stain) were visualized using a light microscope (original magnification ×20).