Figure 5. HCV core protein does not activate the ERN1-XBP1 pathway of UPR. (A) Huh7 cells were transfected with core protein expression plasmids or empty vectors. Cells treated with 300 nM Tg or 10 mM DTT for 16 h before harvesting were used as positive controls. At 48 h post-transfection, the cells were harvested and RT-PCR was performed using XBP1-specific primers. Then the PCR products were digested by Pst I, the unspliced XBP1 products were digested into 295 bp and 147 bp fragments, while the 416 bp spliced XBP1 products could not be digested by Pst I. XBP1(u) represents the unspliced XBP1 PCR products, and XBP1(s) represents the spliced XBP1 PCR products. ACTB was used as the loading control. (B) The cells treated as (A) were analyzed by western blotting using the specific anti-XBP1 antibody to detect the unspliced and spliced XBP1 proteins. (C) Frame diagrams of the reporter plasmid designated Flag-XBP1-FLuc encoding an N-terminal Flag-tagged XBP1 along with the C terminus fused to firefly luciferase (FLuc) at the second ORF of XBP1. (D) Huh7 cells were transfected with core protein expression plasmids or empty vectors along with Flag-XBP1-FLuc and Renilla luciferase pRL-SV40 plasmids, and the cells treated with 300 nM Tg or 10 mM DTT for 16 h before harvesting were used as positive controls. At 48 h post-transfection, the cells were harvested and lysed for measuring XBP1 mRNA splicing. The fold-change is expressed relative to mock-transfected control from at least 3 independent experiments (**P < 0.01). (E) Expression of firefly luciferase (FLuc) and core protein in (D) were analyzed by western blotting using the indicated antibodies. (F) Huh7 cells were transfected with core protein expression plasmids or empty vectors. Cells treated for 12 h with 0.25 μM C42 (11'-deoxyverticillin A), and which exhibited phosphorylation of MAPK9/MAPK8, were used as positive controls. At 48 h post-transfection, the cells were harvested and analyzed by western blotting using the indicated antibodies to measure the phosphorylation of MAPK9/MAPK8.