Activation of the complete IL-2 promoter by bpV compounds.

(A) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg pIL-2–LUC plasmid. After a 24-hour incubation, cells were either left untreated (▪) or were stimulated with PHA (3 μg/mL)/PMA (20 ng/mL) (■) or bpV[pic] (10 μM)/PMA (20 ng/mL) (●) for various times (0, 4, 6, 8, 12, and 24 hours), lysed, and monitored for luciferase activity. (B) J–IL-2–LUC cells were either left untreated (■) or pretreated for 15 minutes with FK506 (10 ng/ml) (▪) before being left untreated or stimulated with PHA (3 μg/mL)/PMA (20 ng/mL) and bpV[pic] (10 μM)/PMA (20 ng/mL). Cells were lysed 8 hours after stimulation, and luciferase activity was monitored. (C) J–IL-2–LUC cells were treated with various activators (PHA, 3 μg/mL; bpV[pic], 10 μM; Iono, 1 μM; PMA, 20 ng/mL; anti-CD3 antibody, 3 μg/mL; anti-CD28 antibody, 1 μg/mL) and lysed after 8 hours to be monitored for luciferase activity. (D) J–IL-2–LUC cells were treated with the same activators as in panel C. Cell-free supernatants were collected 24 hours after stimulation, and IL-2 was quantified through ELISA as described in “Materials and methods.” Luciferase activity was monitored as described in “Materials and methods.” Results for all panels are the means ± SD for triplicate samples and are representative of 2 independent experiments.