1. Volume 137, Issue 5, Pages 1827-1835 (November 2009)
The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2α Via Protein Kinase Activated by Double-Stranded RNA Activation 
Menashe Elazar, Michael Liu, Sean A. McKenna, Ping Liu, Elizabeth A. Gehrig, Joseph D. Puglisi, Jean–François Rossignol, Jeffrey S. Glenn 
Gastroenterology 
Volume 137, Issue 5, Pages (November 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
NTZ increases eIF2α phosphorylation. (A) Subgenomic replicon cells (RP7), (B) full-length replicon cells (FLRP1), and (C) Huh-7.5 cells infected with HCVcc virus (4 weeks before treatment). The cells were treated with the indicated NTZ concentrations at a final concentration of 0.5% dimethyl sulfoxide for 72 hours. Seventy-two hours after treatment the cells were harvested and equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot for total and phosphorylated eIF2α. The relative amount of phosphorylated eIF2α in each lane was normalized to total eIF2α and expressed relative to the nontreated sample. Huh-7.5 cells stably carrying the (D) ATF-4 luciferase reporter or the (E) XBP/Ire1 luciferase reporter plasmids were infected with HCVcc and treated with NTZ for 24 hours as described in the Material and Methods section. As control, 1 μmol/L thapsigargin (tg) was added to noninfected cells for 12 hours before cell harvest and results were analyzed as the fold increase of luciferase activity relative to nontreated noninfected (nt/ni) cells. The mean and standard deviation are presented (n = 7).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
NTZ-induced eIF2α phosphorylation is greater in the presence of IFN. (A) Subgenomic replicon cells (RP7) were plated and treated as described in the Materials and Methods section in the presence or absence of 3 IU/mL IFN for 72 hours. The cells were harvested and analyzed as described in the Figure 1 legend. One micromolar of thapsigargin (tg) was added for 12 hours as a positive control for induction of eIF2α phosphorylation. (B) Huh-7.5 cells stably carrying the ATF-4 luciferase reporter plasmid were infected with HCVcc and treated with NTZ for 24 hours with or without 50 IU/mL IFN for the last 4 hours of incubation. As control, 1 μmol/L Tg was added to noninfected cells for 12 hours before cell harvest. The cells were harvested as described in the Materials and Methods section, and results were analyzed as the fold increase of ATF-4 luciferase activity relative to nontreated cells. The mean and standard deviation are presented (n = 7).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
NTZ increases PKR phosphorylation. Full-length HCV replicon cells were plated, treated with NTZ in the presence or absence of IFN, and harvested as described in the Materials and Methods section. The cell lysates were analyzed for the level of phosphorylated PKR, total PKR, and actin. The relative amounts of phosphorylated PKR were normalized to actin and expressed relative to the nontreated samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
NTZ increases PKR autophosphorylation in vitro. (A) Purified PKR (0.3 μmol/L) was incubated with HIV-TAR RNA activator (0.3 μmol/L) and N-eIF2α (2 μmol/L) in the presence of 32P-γATP and increasing amounts of NTZ. (B) Purified PKR (0.3 μmol/L) was incubated in the presence (top panel) or absence (bottom panel) of HIV-TAR RNA activator (0.3 μmol/L) in the presence of 32P-γATP and increasing amounts of NTZ.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
NTZ's effect on eIF2α phosphorylation is mediated via PKR activation. (A) Huh-7.5 cells were transfected with mock or HCV J6/JFH RNA with or without PKR inhibitor (VA1 RNA). Two hours after transfection, the cells were treated with NTZ or 0.5% dimethyl sulfoxide for 24 hours. Cells were harvested as described in the Figure 1 legend and the resulting lysates were analyzed for phosphorylated and total eIF2α. The relative amounts of phosphorylated eIF2α were normalized to total eIF2α and expressed relative to the nontreated sample. The figure represents at least 2 independent experiments. (B) Huh-7.5 cells were cotransfected with the ATF-4 luciferase reporter plasmid, a cytomegalovirus promoter–driven renilla luciferase expression plasmid, and a plasmid expressing either wild-type PKR or a dominant-negative form of PKR (K296R). Twenty-four hours after transfection the cells were infected with HCVcc and 2 hours after infection the cells were treated with NTZ as indicated for an additional 24 hours. The cells were harvested as described in the Materials and Methods section. The results were normalized to the renilla luciferase activity and presented as the fold increase of firefly luciferase activity relative to nontreated, PKR transfected cells. The mean and standard deviation of at least 3 transfections is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions