Nfv does not alter protein or mRNA expression or caspase activity.

(A) Apoptosis was induced by stimulation with 10 μM CPT and assessed after annexin-V and propidium iodide staining by flow cytometry (data representative of 3 independent experiments). (B) Immunoblots of pro–caspase-3 and -8 from whole-cell lysates (40 μg of protein per lane) of Jurkat cells treated overnight with MeOH, 10 μM AZT, or 7 μM Nfv. The PCNA immunoblot confirms equal loading. Jurkat control cells (left panel) or cells pretreated for 1 hour with 10 μg/mL CHX (right panel) before overnight culture with MeOH (▪) or 7 μM Nfv (░). (C) RPA of mRNA (40 μg per lane) from Jurkat cells treated for 3 days with MeOH, 10 μM AZT, or 7 μM Nfv. P indicates probe; C, MeOH; A, AZT; N, Nfv; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Active human recombinant caspase-3 was incubated with its fluorogenic substrate (DEVD-AFC) alone (■) or in the presence of 100 μM DEVD-FMK (♦), 7 μM Nfv (▴), or MeOH (○), and the kinetics were assessed over 30 minutes (top panel). Similar results were also obtained with active human recombinant caspase-1, -6, -7, and -8 (data not shown). Data are representative of at least 3 independent experiments for each caspase tested. HIV-1 protease cleavage of gag/pol fluorogenic substrate was assessed alone (■), with 7 μM Nfv (▴), or with methanol (○) (bottom panel).