2-OH-E+, 2-hydroxyethidium; ATP, adenosine triphosphate; cDNA, complementary DNA; DPI, diphenylene iodonium; Duox, dual oxidase; GAPDH, glyceraldehyde

3-phosphate dehydrogenase; GSH, glutathione; H2O2, hydrogen peroxide; HCV, hepatitis C virus; HDAC1, histone deacetylase 1; HE, dihydroethidium; HPLC, high-performance liquid chromatography; IgG, immunoglobulin phosphatase inhibitor library G; JFH1, Japanese fulminant hepatitis 1; L-NMA, NG-methyl-L-arginine acetate; mRNA, messenger RNA; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Nox, NAD(P)H oxidase; PI, propidium iodide; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; Autophagy inhibitor order ROS, reactive oxygen species; rRNA, ribosomal RNA; siRNA, small interfering

RNA; SOD, superoxide dismutase; TGFβ, transforming growth factor beta. pJFH1 (which generates infectious virus particles of genotype 2a), its replicative-null mutant (pJFH1-GND), subgenomic pSgJFH1-Luc (which supports viral RNA genome replication without generating virus particles), and genotype 1b pEF-CG1bRbz/Neo plus its replicative-null mutant (pEF-CG1b GNDRbz/Neo) were used (Sg and Luc indicate subgenomic and luciferase, respectively. EF denotes elongation factor 1α promotor. Rbz and Neo indicate ribozyme and neomycin phosphotransferase, respectively).10, 11 Huh7 human hepatoma cells were transfected with in vitro transcribed HCV RNA and cultured, as previously described.12 Telomerase-reconstituted primary human hepatocytes

were transfected with pEF-CG1bRbz/Neo, pEF-CG1bGNDRbz/Neo, or control EF-driven vector alone as described for Huh7, and cell clones that were stably transfected with these constructs were selected and maintained in a G418-containing medium (Invitrogen).13 For virus infection, 2 mL of the extracellular medium from JFH1-transfected cells was used to inoculate naïve Huh7 cells with 3 mL of fresh medium, as described.10 Then, the cells were cultured and harvested at various time points, as indicated in the Results section. Huh7-Nox4 cells, which learn more constitutively overexpress Nox4 enzyme, were generated by the transfection of Huh7 cells with human Nox4 complementary DNA (cDNA) with Lipofectamine 2000 (Invitrogen) and by the selection of stable cell clones with 0.5 mg/mL G418 (Invitrogen). Control cell clones (Huh7-pcDNA), transfected with an empty plasmid vector alone, were also generated. These cells were maintained in a 0.25 mg/mL G418-containing medium. For experiments involving these or other stable cell clones, G418 was removed from the cell culture medium 1 day prior to the experiment. HCV-infected and uninfected human liver tissues were acquired through the National Disease Research Interchange.