Evaluation of BB-1 in other human leukemia cell lines. ST1, U937, K562, Ramos, and HL60 cells (10⁶ cells/mL) were incubated for 48 hours with 0.2 μg/mL TRAIL [T], 12.5 μg/mL BB-1 [B], BB-1+TRAIL (simultaneous addition [B+T]), or BB-1+TRAIL (0.5 μg/mL TRAIL was added after incubation with 12.5 μg/mL BB-1 for 24 hours [B→T]), or without reagents (C). (A) Sensitivity. Cell proliferation was assessed by MTS assay, and results are expressed relative to the value for the control cells cultured without inducers. Data are the mean ± SD from 3 independent experiments. (B,D-E) Evaluation of the molecules involved in the apoptotic pathway. Cells were harvested at the indicated time points, Western blotting was performed using 30 μg cell extract, and each molecule was detected with the antibodies described in “Materials and methods.” (C) Mitochondrial membrane potential (ΔΨm) and cytochrome c translocation. After treatment, cells were evaluated using DiOC6 by FCM, and loss of ΔΨm (%) is indicated. Data are the mean ± SD of 3 independent experiments. Using 10 μg cytosolic extract, cytochrome c was detected by Western blotting as described in “Materials and methods.”