Wogonin-induced apoptosis is caspase-dependent but death receptor–independent. (A) Wogonin-induced apoptosis involves caspases. CEM cells were treated with 25 μM Wogonin in the presence or absence of different doses of the pan-caspase inhibitor zVAD-fmk for 24 hours. Apoptotic cells were analyzed by FSC/SSC. (B) The death receptor system is not required for Wogonin-induced apoptosis. FADD-deficient (FADDdef) and parental (A3) Jurkat cells were treated with different doses of Wogonin for 24 hours. (C) Wogonin-induced apoptosis does not require newly synthesized proteins. CEM cells were treated with 25 μM Wogonin in the presence or absence of cycloheximide (chx) (1 μg/mL). (D) Wogonin does not activate caspase-8 in vitro. Cell lysates from Jurkat cells were subjected to an in vitro caspase activity assay with or without 50 μM Wogonin. Results are the average of 2 assays in triplicate measurements. (E) Overexpression of Bcl-2 prevents Wogonin-induced apoptosis. Jurkat cells stably expressing Bcl-2 and control Jurkat cells (neo) were treated with different doses of Wogonin for 24 hours. Apoptotic cells were analyzed by FSC/SSC in triplicates. (F,G) Wogonin induces depolarization of the mitochondrial membrane potential. Jurkat and CEM cells were treated with 100 μM Wogonin for different times as indicated, and the mitochondrial membrane potential was determined by FACS. (H) Wogonin induces activation of caspase-9 and -3. Jurkat T cells were treated with 100 μM Wogonin for 8 hours in the absence or presence of 20 μg/mL caspase-9 inhibitor z-LEHD-fmk. Cell lysates were subjected to Western blot analysis with specific antibodies as indicated. (I) Jurkat T cells were treated with 100 μM Wogonin for 36 hours in the absence or presence of different amounts of z-LEHD-fmk. Apoptotic cell death was determined by FACS for DNA fragmentation. Error bars represent SD.