Fig. 7.

La-DN inhibits H₂O₂-activated translation ex vivo. (a) Relative translational activities in Huh7 cells transfected with either the dominant-negative La (La-DN) mutant or the control vector for 24 h, split into a 24-well plate at a density of 1.2×10⁵ for 24 h before being transfected with the bicistronic pRL1b RNA transcript for 4 h followed by treatment with the indicated doses of H₂O₂ for 4 h. The HCV IRES and cap-translational activities were measured by firefly and Renilla luciferase activities, normalized with respect to total protein, and expressed relative to their respective 0 µM H₂O₂ controls, which are set as 1. The IRES/cap ratio is represented by the ratio of firefly to Renilla luciferase activities and is expressed relative to their respective 0 µM H₂O₂ control, which is set as 1. The values obtained represent the mean±sem of three independent experiments, performed in triplicates. RLU, Relative luciferase units. (b) Western blots showing expression of La-DN (detected by the myc-tag antibody clone A-14 Santa Cruz 1 : 1000; HRP-conjugated anti-rabbit antibody, 1 : 1000) at 48 h post-transfection and the internal loading control β-tubulin (1 : 5000; HRP-conjugated anti-mouse antibody 1 : 1000). (c) A representation of three independent dichlorofluorescin fluorometric assays, performed in triplicates, showing the kinetics of ROS generation in plasmid-transfected Huh7 cells (1.2×10⁵ per well in a 24-well plate) after treatment with doses of H₂O₂, as indicated. FL, Fluorescence units. (d) XTT assay showing viability of plasmid-transfected Huh7 cells (1.2×10⁵ per well in a 24-well plate) after treatment with doses of H₂O₂, as indicated, for 24 h. The values obtained represent the mean±sem of three independent experiments, performed in triplicates, and are expressed relative to the respective 0 µM H₂O₂ controls, which are set as 100 %. Significance of the difference *P<0.05.