Ca²⁺ is involved in Wogonin-induced apoptosis. (A,B) Wogonin mobilizes intracellular Ca²⁺ in malignant but not in normal T cells. Jurkat, CEM, naive (day 0), and effector (day 6) normal T cells were treated in PBS with either 0.5 μM ionomycin or 50 μM Wogonin as indicated. The intracellular Ca²⁺ release was monitored by FACS. Results are representative of 3 independent experiments. (C) The Ca²⁺ influx antagonist Nifedipine prevents Wogonin-induced apoptosis. CEM cells were treated with 100 μM Wogonin in the presence or absence of different amounts of nifedipine (added 2 hours before Wogonin treatment) for 24 hours. Apoptotic cell death was analyzed by FACS for DNA fragmentation. Results are representative of 3 independent experiments in triplicate measurements. (D) The calcineurin inhibitor FK506 inhibits Wogonin-induced apoptosis. CEM cells were treated with 50 to 100 μM Wogonin in the presence or absence of different amounts of FK506 (added 2 hours before Wogonin treatment) for 24 hours. Apoptotic cell death was analyzed by FACS for DNA fragmentation. Results are representative of 2 independent experiments in triplicate measurements. (E) CEM cells were treated with 50 μM Wogonin in the presence or absence of different concentrations of CsA (preincubated for 2 hours before Wogonin treatment) for 24 hours. Apoptotic cell death was analyzed by a decrease in FSC/SSC. Results are representative of 3 independent experiments. (F) Effects of the mitochondrial uniporter antagonist RU-360 on Wogonin-induced apoptosis. CEM cells were treated with Wogonin in the presence or absence of RU-306 for 24 hours. Apoptotic cell death was analyzed by FACS for DNA fragmentation. Error bars represent SD.