Effect of NFV on apoptotic signaling events in vitro and in vivo. (A) Jurkat T cells were stimulated with agonistic anti-Fas antibody (CH-11) in the presence or absence of varying concentrations of PI and analyzed for annexin positivity. *P < 0.05. (B) Western blot analysis of the Fas signaling events of caspase-8, -9, and -3 cleavage, BID cleavage, or cytosolic translocation of cytochrome c (cyt c) in Jurkat T cells treated or not treated with CH-11 with or without NFV. tBID, truncated BID. (C) Caspase-8 and caspase-3 activity was also assessed in Jurkat T cells stimulated with CH-11 in the presence or absence of NFV. ^#P < 0.01. (D) Jurkat cells were transiently transfected by a GFP plasmid or a caspase-9 GFP plasmid, treated or not treated with 7 μM NFV or 100 μM LEHD, cultured for 6 hours, stained with PI, and analyzed by cytofluorometry for hypoploidy. (E) Jurkat cells stimulated with CH-11 in the presence or absence of NFV were analyzed for loss of DiOC₆ retention. Hepatocytes isolated from mice receiving Jo-2 with NFV/RIT or control (as in Figure 1) were also analyzed for caspase-8 and caspase-3 activity (F) and loss of DiOC₆ retention (G). ^†P < 0.005.