(A) Dose-dependent inhibition of protein synthesis in vivo by CBFs. HeLa cells were incubated with the indicated concentrations of FA and silvestrol for 1 hour, with [³⁵S]methionine added 10 minutes before the end of the incubation. The rate of [³⁵S]methionine (³⁵S]-Met) incorporation is expressed relative to that of cells treated with vehicle (MeOH). Results are expressed as mean ± SEM of 2 experiments. (B) CBFs primarily impact protein synthesis in vivo. HeLa cells were incubated with 5 μM FA, 0.4 μM silvestrol, or vehicle (MeOH) for 1 hour. The rate of incorporation of each radioisotope tracer into TCA-insoluble material is expressed relative to that in MeOH-treated cells. Results are expressed as mean ± SEM of 2 experiments. (C) Inhibition of translation by CBFs is reversible. HeLa cells were incubated for 1 hour with 10 μM anisomycin, 5 μM FA, 0.4 μM silvestrol, or MeOH. Cells were then washed with PBS and incubated with medium lacking compound for the indicated times. Ten minutes before harvesting, [³⁵S]methionine was added to the culture. The rate of [³⁵S]methionine incorporation into TCA-insoluble material is expressed relative to that in MeOH-treated cells. Results are expressed as mean ± SEM of 3 experiments. Anisomycin acts a positive control, since recovery of protein synthesis from inhibition with this compound occurs within an hour of its removal from cells (65). (D) CBFs inhibit cap-dependent translation in vivo. Top: Schematic representation of pcDNA/Ren/HCV/FF expression vector. Bottom: Effect of FA on cap-dependent and HCV IRES–mediated translation in 293 cells transfected with pcDNA/Ren/HCV/FF. Luciferase activity is expressed relative to that in MeOH-treated cells and is the mean ± SEM of 2 experiments. (E) Silvestrol does not induce eIF2α phosphorylation. HeLa cells were incubated for 2 hours in the presence of vehicle (DMSO), thapsigargin (2 μg/ml), or silvestrol (400 nM), after which extracts were analyzed by Western blotting. (F) Silvestrol induces apoptosis at concentrations higher than those required to inhibit protein synthesis. Jurkat cells were incubated with the indicated silvestrol concentrations for 13 hours, after which the rate of [³⁵S]methionine incorporation or the percentage of living cells was measured. Results are expressed as mean ± SEM of 2 experiments.