Tunicamycin induces apoptosis and cell-cycle arrest. (A) BCWM.1 cells were cultured with tunicamycin (0.01 μg/mL to 5 μg/mL) for 48 hours. Then the percentage of cells undergoing apoptosis was studied using positivity of Apo2.7 staining by flow cytometry. (B) Tunicamycin induces BCWM.1 WM cell apoptosis at 10 hours. BCWM.1 cells were cultured with tunicamycin (0.01 μg/mL to 5 μg/mL) then whole-cell lysates were subjecting to Western blotting using anti–caspase-9, –caspase-8, –caspase-3, -PARP, Mcl-1, p-Bcl2, and α-tubulin antibodies. (C) BCWM.1 cells were cultured without (control) and with tunicamycin (0.5 μg/mL) for 24 hours. Cell cycle was then studied using PI staining by flow cytometry. Percentages indicate cells in sub-G1 phase, G1 phase, and G2/M phase for 0.5 μg/mL tunicamycin. (D) BCWM.1 cells were cultured for 6 hours. Whole-cell lysates were subjecting to Western blotting using anti-CDK4, -CDK6, -CDK2, –p-CDK2, -p27, -p21, -p53, –p-Rb, and α-tubulin antibodies. All results represent mean plus or minus SD of triplicate experiments (A,C).