The role of caspase-8 and FADD in NPI-0052–induced apoptosis. (A) Caspase-8 and FADD expression. Jurkat, I2.1 and I9.2 protein lysates were assessed for expression of caspase-8 and FADD by immunoblotting. (B) NPI-0052–induced caspase-3 activity is mediated by caspase-8 and FADD. Jurkat, I2.1 (FADD deficient) and I9.2 (caspase-8 deficient) cells were incubated in the presence of 200 nM NPI-0052 for 6 hours, and caspase-3 activity was monitored by cleavage of DEVD-amc. Anti-Fas antibody (CH-11) was used as positive control. Values represent the mean ± SD from 3 separate studies performed in triplicate (P > .05). (C) Re-expression of FADD in I2.1 cells induces caspase-3 activation. I2.1 cells and I2.1 transfected cells with full-length FADD (I2.1-FADD GFP) were treated with NPI-0052 for 6 hours, and caspase-3 activity was monitored. *P > .05. Inset: PI staining assessed DNA fragmentation in I2.1 and I2.1-FADD GFP cells exposed to 200 ng/mL CH-11 for 12 hours. (D) FADD and caspase-8 are required for NPI-0052–induced mitochondrial injury. Following 4 hours of 200 nM NPI-0052 exposure, TMRE staining, followed by FACS analysis, was employed to monitor Jurkat, I2.1 and I9.2 cells for mitochondrial injury. Histograms shown are representative of 3 separate experiments.