Perifosine and bortezomib-induced apoptosis and cell-cycle arrest were measured in WM cells. (A) BCWM.1 cells were cultured with either perifosine (5 μM and 10 μM), bortezomib (5 nM and 10 nM), and the combination for 48 hours. Then the percentage of cells undergoing apoptosis was studied using Apo2.7 staining and flow cytometry. (B) BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (5 nM and 10 nM), or the combination for 10 hours. Whole cell lysates were subjected to Western blotting using anticaspase 9, -caspase 8, -caspase 3, -PARP, -MCL1, Bcl-xl, and –α-tubulin antibodies. (C) Cell cycle was then studied using PI staining by flow cytometry. BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (10 nM), or the combination for 24 hours. Percentages indicate cells in sub-G₁ phase, G₀/G₁ phase, and G₂/M phase. (D) BCWM.1 cells were cultured with either perifosine (10 μM), bortezomib (5 nM and 10 nM), or the combination for 12 hours. Whole cell lysates were subjecting to Western blotting using anti-CDK4, -CDK6, -CDK2, -p-CDK2, -p27kip1, -p21Waf1/Cip1, -p53, -p-Rb, and –α-tubulin antibodies. Data represent mean plus or minus SD of triplicate experiment (A,C).