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. 2016 Jul 27;90(16):7231–7247. doi: 10.1128/JVI.00326-16

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Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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FIG 3 — TRIB3 negatively regulates HCV propagation. (A, top) Schematic illustration of the experimental design. (Middle) Huh7 cells were transfected with 20 nM the indicated siRNAs and then infected with Jc1 48 h after transfection. At 2 days postinfection, both intracellular TRIB3 and HCV RNA levels were analyzed by qRT-PCR. (Bottom) Using the same cell lysates, protein levels were determined by immunoblot analysis with the indicated antibodies. Band intensities of normalized TRIB3 and HCV proteins were analyzed by using ImageJ. (B) Huh7 cells were transfected with either negative-control siRNA or increasing concentrations of TRIB3-specific siRNAs for 48 h and then infected with Jc1 for 48 h. Both intracellular TRIB3 and HCV RNA levels were analyzed by qRT-PCR. (C, top) Schematic illustration of the experimental design. Huh7 cells were transfected with either the vector or Flag-tagged TRIB3 for 24 h, followed by Jc1 infection. (Bottom) At 2 days postinfection, the intracellular HCV RNA level was analyzed by qRT-PCR (left), and protein levels were determined by immunoblot analysis using the indicated antibodies (right). Band intensities of normalized HCV proteins were analyzed by using ImageJ. (D) Huh7 cells were transfected with either the vector or increasing amounts of Flag-tagged TRIB3 for 24 h and then infected with Jc1. At 48 h postinfection, intracellular HCV RNA levels were determined by qRT-PCR. (E) Huh7 cells were transfected with 20 nM negative-control or TRIB3-specific siRNAs. Ninety-six hours after transfection, cell viability was determined by a WST assay. (F, top) Schematic diagram of plasmid constructs. UTR, untranslated region. (Middle) Schematic illustration of the experimental design. (Bottom) Huh7 cells were transfected with 20 nM the indicated siRNAs. Twenty-four hours after transfection, cells were further transfected with either the empty vector, wild-type (WT) TRIB3, or an Akt binding-defective TRIB3 mutant (ΔAkt mutant TRIB3) plasmid, respectively, for 24 h, followed by Jc1 infection. At 48 h postinfection, intracellular HCV RNA levels were analyzed by qRT-PCR. The asterisks indicate significant differences (**, P < 0.01). Experiments were performed in triplicate. (G) HCV subgenomic replicon cells were transfected with either negative-control siRNA or increasing amounts of TRIB3-specific siRNAs for 72 h. Intracellular HCV RNA levels were analyzed by qRT-PCR. (H) Huh7 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were further cotransfected with the pRL-HL dual-reporter plasmid and the pCH110 β-galactosidase plasmid. Forty-eight hours after transfection, relative luciferase activities were determined. (I, top) Schematic illustration of the experimental design. (Middle) Huh7 cells were infected with Jc1 for 3 days and then transfected with the indicated siRNAs for 48 h. Intracellular HCV RNA levels were analyzed by qRT-PCR. (Bottom) Naive Huh7 cells were infected with virus-containing culture supernatants harvested from the cells described above. Intracellular HCV RNA levels were determined by qRT-PCR. (J) Huh7 cells were transfected with the indicated siRNAs for 48 h. Cells were then infected with either HCVpp derived from genotype 2a (JFH1) or genotype 1a (H77) or VSVpp. At 48 h postinfection, cells were harvested, and viral entry was determined by luciferase activity. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Experiments were performed in triplicate. Neg denotes universal control siRNA. RLU, relative light units.

FIG 3 — TRIB3 negatively regulates HCV propagation. (A, top) Schematic illustration of the experimental design. (Middle) Huh7 cells were transfected with 20 nM the indicated siRNAs and then infected with Jc1 48 h after transfection. At 2 days postinfection, both intracellular TRIB3 and HCV RNA levels were analyzed by qRT-PCR. (Bottom) Using the same cell lysates, protein levels were determined by immunoblot analysis with the indicated antibodies. Band intensities of normalized TRIB3 and HCV proteins were analyzed by using ImageJ. (B) Huh7 cells were transfected with either negative-control siRNA or increasing concentrations of TRIB3-specific siRNAs for 48 h and then infected with Jc1 for 48 h. Both intracellular TRIB3 and HCV RNA levels were analyzed by qRT-PCR. (C, top) Schematic illustration of the experimental design. Huh7 cells were transfected with either the vector or Flag-tagged TRIB3 for 24 h, followed by Jc1 infection. (Bottom) At 2 days postinfection, the intracellular HCV RNA level was analyzed by qRT-PCR (left), and protein levels were determined by immunoblot analysis using the indicated antibodies (right). Band intensities of normalized HCV proteins were analyzed by using ImageJ. (D) Huh7 cells were transfected with either the vector or increasing amounts of Flag-tagged TRIB3 for 24 h and then infected with Jc1. At 48 h postinfection, intracellular HCV RNA levels were determined by qRT-PCR. (E) Huh7 cells were transfected with 20 nM negative-control or TRIB3-specific siRNAs. Ninety-six hours after transfection, cell viability was determined by a WST assay. (F, top) Schematic diagram of plasmid constructs. UTR, untranslated region. (Middle) Schematic illustration of the experimental design. (Bottom) Huh7 cells were transfected with 20 nM the indicated siRNAs. Twenty-four hours after transfection, cells were further transfected with either the empty vector, wild-type (WT) TRIB3, or an Akt binding-defective TRIB3 mutant (ΔAkt mutant TRIB3) plasmid, respectively, for 24 h, followed by Jc1 infection. At 48 h postinfection, intracellular HCV RNA levels were analyzed by qRT-PCR. The asterisks indicate significant differences (**, P < 0.01). Experiments were performed in triplicate. (G) HCV subgenomic replicon cells were transfected with either negative-control siRNA or increasing amounts of TRIB3-specific siRNAs for 72 h. Intracellular HCV RNA levels were analyzed by qRT-PCR. (H) Huh7 cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were further cotransfected with the pRL-HL dual-reporter plasmid and the pCH110 β-galactosidase plasmid. Forty-eight hours after transfection, relative luciferase activities were determined. (I, top) Schematic illustration of the experimental design. (Middle) Huh7 cells were infected with Jc1 for 3 days and then transfected with the indicated siRNAs for 48 h. Intracellular HCV RNA levels were analyzed by qRT-PCR. (Bottom) Naive Huh7 cells were infected with virus-containing culture supernatants harvested from the cells described above. Intracellular HCV RNA levels were determined by qRT-PCR. (J) Huh7 cells were transfected with the indicated siRNAs for 48 h. Cells were then infected with either HCVpp derived from genotype 2a (JFH1) or genotype 1a (H77) or VSVpp. At 48 h postinfection, cells were harvested, and viral entry was determined by luciferase activity. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Experiments were performed in triplicate. Neg denotes universal control siRNA. RLU, relative light units.