The Roles of Endoplasmic Reticulum Overload Response Induced by HCV and NS4B Protein in Human Hepatocyte Viability and Virus Replication
Fig 3
Activation of cancer-related NF-κB signaling pathway by HCV via EOR in human hepatoma cells.
(A). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 0, 0.02, 1 and 5. At 48 h postinfection, cells were subjected to indirect immunofluorescece with mouse anti-Core antibody and Alexa FluorR 555 anti-mouse secondary antibody. Nuclei were stained with DAPI. (B). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 0, 0.02, 1 and 5, and transfected with plasmids consisting of NF-κB-Luc and pRL-CMV. After 48 h, cells were subjected to luciferase assay for NF-κB activation. Values are means ± SD (n = 3). * P < 0.05. Scale bars represent 50 μm. (C) and (D). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 5, and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. (C). Western blot analysis of protein levels of C-myc, Mcl-1, Cyclin D1, MMP-9, phospho-IκBα, NS4B and actin in cells at 48 h posttransfection. Actin protein bands act as internal control. p50 protein accumulation in the nuclear extracts was also analyzed by western blot. YY1 acts as a nuclear-specific control. (D). Real-time RT-PCR analysis of C-myc, Mcl-1, Cyclin D1 and MMP-9. GAPDH act as internal control. Values are means ± SD (n = 3). * P < 0.05. (E). Primary human hepatocytes in 24-well plate were infected with JFH1 at a virus titer (IU/cell) of 1. Mock-infected primary human hepatocytes were used as controls. At 48 h postinfection, Core, NS4B and actin proteins were determined by Western blot. (F). Primary human hepatocytes in 96-well plates were infected with JFH1 at a virus titer (IU/cell) of 1, and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h postinfection, transcripts of C-myc, Mcl-1, Cyclin D1 and MMP-9 in cells were analyzed by real-time RT-PCR. GAPDH acts as internal control. Values are means ± SD (n = 3). * P < 0.05. (G). Mock-infected primary human hepatocytes in 96-well plates and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h postinfection, transcripts of C-myc, Mcl-1, Cyclin D1 and MMP-9 in cells were analyzed by real-time RT-PCR. GAPDH acts as internal control. Values are means ± SD (n = 3). * P < 0.05. (H). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 5, transfected with plasmids consisting of NF-κB-Luc and pRL-CMV, and treated with SN50 (40 μM) for 4 h, Ryanodine (100 nM) for 4 h, Ruthenium red (50 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h posttranfection, cells were subjected to luciferase assay. Values are means ± SD (n = 3). * P < 0.05.