![Fig. 2. NFAT-driven luciferase gene expression is induced by bpV molecules and is FK506 sensitive. / (A) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg pNFAT-LUC. Following a 24-hour incubation period, cells were either left untreated (▪) or stimulated with PHA (3 μg/mL)/PMA (20 ng/mL) (■) or bpV[pic] (10 μM) (●) and lysed after 4, 6, 8, 12, and 24 hours of stimulation. (B) Ficoll Hypaque–isolated PBMCs were first treated with PHA-L for 20 hours and then electroporated in the presence of 15 μg of pNFAT-LUC. Following a 2-hour incubation period, cells were stimulated with bpV[pic] (10 μM) (⋄) or PMA (20 ng/mL)/Iono (1 μM) (■) for 8 hours. (C) Jurkat cells stably transfected with pNFAT-LUC were either left untreated (■) or pretreated with FK506 (10 ng/mL) (▪). After 15 minutes, cells were stimulated for 8 hours with PHA (3 μg/mL), PMA (20 ng/mL), PHA (3 μg/mL)/PMA (20 ng/mL), bpV[pic] (10 μM), or bpV[pic] (10 μM)/PMA (20 ng/mL). (D) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg of pNFAT-LUC plus 15 μg of either the control vector pEGFP (■ or the NFAT-inhibitor expression vector pVIVIT-GFP (▪). Following a 24-hour incubation period, cells were left untreated or were stimulated with either PHA (3 μg/mL), PMA (20 ng/mL)/Iono (1 μM), or bpV[pic] (10 μM) for 8 hours. Cells were lysed and evaluated for luciferase activity. Values are the mean of 3 different measured samples ± SD. This is representative of 2 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/97/8/10.1182_blood.v97.8.2390/5/h80810920002.jpeg?Expires=1722696753&Signature=dtFwmAmw758LAT5mP5t6BuDFHwYX1JXrZ75TP1x~AKHrXHMpBxS6du~BhKWv0caAH-P~PtbLRUhIiCEJmLHs1KRlTcikkXgv~7Cg9NW7dE5tuUMK8xfWeiPR9KQmC6ekroEBO9999kF8iN1TfuzlMgy08pf-5XIGwkyQWts9ZtqFKXmTKy-sHKoyD~16t6tMcwJ60VGxSeEJdziXSB--JaihNTyQ~RLYx4xOT33sV0OyZpvfcAG7MyJq5~R340bvJHJZDc7sUIifNDBQxCI~oe1tPiw~bKToNkw17Pwt5bT1aJHa9oAtxto2JkqDm-CxcO9pIUMR9DuBnrmCzPn0zQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
NFAT-driven luciferase gene expression is induced by bpV molecules and is FK506 sensitive.
(A) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg pNFAT-LUC. Following a 24-hour incubation period, cells were either left untreated (▪) or stimulated with PHA (3 μg/mL)/PMA (20 ng/mL) (■) or bpV[pic] (10 μM) (●) and lysed after 4, 6, 8, 12, and 24 hours of stimulation. (B) Ficoll Hypaque–isolated PBMCs were first treated with PHA-L for 20 hours and then electroporated in the presence of 15 μg of pNFAT-LUC. Following a 2-hour incubation period, cells were stimulated with bpV[pic] (10 μM) (⋄) or PMA (20 ng/mL)/Iono (1 μM) (■) for 8 hours. (C) Jurkat cells stably transfected with pNFAT-LUC were either left untreated (■) or pretreated with FK506 (10 ng/mL) (▪). After 15 minutes, cells were stimulated for 8 hours with PHA (3 μg/mL), PMA (20 ng/mL), PHA (3 μg/mL)/PMA (20 ng/mL), bpV[pic] (10 μM), or bpV[pic] (10 μM)/PMA (20 ng/mL). (D) Jurkat cells were transiently transfected by the DEAE-Dextran protocol with 15 μg of pNFAT-LUC plus 15 μg of either the control vector pEGFP (■ or the NFAT-inhibitor expression vector pVIVIT-GFP (▪). Following a 24-hour incubation period, cells were left untreated or were stimulated with either PHA (3 μg/mL), PMA (20 ng/mL)/Iono (1 μM), or bpV[pic] (10 μM) for 8 hours. Cells were lysed and evaluated for luciferase activity. Values are the mean of 3 different measured samples ± SD. This is representative of 2 independent experiments.
