Increase of TRAIL-R2 expression by BB-1. (A) Cell surface TRAIL receptors. U937, K562, and HL60 (10⁶ cells/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours, and the cells were stained with antibodies to TRAIL receptors or with an isotype-matched antibody. Cells (10⁴) were counted and analyzed by FCM. Shaded peaks, solid lines, and dotted lines correspond to treated, untreated, and control staining, respectively. RFI was determined as the ratio of mean fluorescence intensity for specific staining to that for control staining. (B) TRAIL receptor mRNA. KOB cells (5 × 10⁵/mL), U937 cells (10⁶ cells/mL), and HL60 cells (10⁶ cells/mL) were incubated with or without 12.5 μg/mL BB-1 for 24 hours. Cells were then harvested, cDNAwas constructed from 1 μg total RNA, and RT-PCR (29 cycles) was performed. mRNA expression of TRAIL-R1 and -R2 in untreated and BB-1–treated cells is shown with that of GAPDH. (C) Correlation between receptor expression and activity of AKT and ERK. KOB cells (5 × 10⁵/mL) or cells (10⁶/mL) from the U937 and K562 lines were treated with the indicated concentration of LY294002 or U0126 for 24 hours and were harvested. The phosphorylation status of AKT and ERK was evaluated by Western blotting, and the cell surface expression of TRAIL-R1 and -R2 was examined by FCM as described for panel A.