Fig. 8.

La-DN inhibits H₂O₂-activated translation in a sub-genomic replicon cell line. (a) Diagrammatic representation of the sub-genomic replicon used in this study. The sub-genomic replicon consists of a bicistronic transcript in which expression of the core (1–15 aa)-neomycin phosphotransferase (C-neo) fusion protein is under the translational control of the HCV IRES element, whereas expression of the HCV non-structural (NS) 3-NS5B polyprotein is under the translational control of the EMCV IRES element. (b) Western blots showing the levels of the C-neo/neo proteins, NS5A, myc-tag dominant-negative La (La-DN) mutant protein and the internal loading control β-tubulin in whole-cell lysates of Huh8 cells transfected with either the empty vector or a vector expressing the La-DN mutant protein and treated with doses of H₂O₂, as indicated, for 1 h. The following concentrations were used: (anti-neo antibody clone 4B4D1, Sigma, 1 : 1000; anti-mouse HRP 1 : 500); (NS5A clone 9E10 1 : 50; anti-mouse HRP 1 : 1000); (anti-myc clone A-14, Santa Cruz 1 : 1000; anti-rabbit HRP 1 : 1000); (anti-β-tubulin clone TUB2.1, Sigma 1 : 1000; anti-mouse HRP 1 : 1000). The levels of C-neo, neo and NS5A were quantified using ImageJ, normalized against β-tubulin and expressed as fold increase relative to the 0 µM H₂O₂ vector control. (c) Agarose gel electrophoresis showing the amplified fragments from RT-PCR using primers specific for the HCV IRES element and the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Huh8 cells transfected with either the empty vector or a vector expressing the La-DN mutant protein and treated with doses of H₂O₂, as indicated, for 1 h. –ve RT, Negative control for the RT reaction; –ve PCR, negative control for the PCR. The bottom panel shows equal quantity of RNA had been used in the RT-PCR. The bands are the 28S and 18S ribosomal RNAs. (d) XTT assay showing viability of Huh8 cells (16 000 per well/96-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 quadruplicates, and are expressed relative to the untreated control, which is set as 100 %. Significance of the difference *P<0.05. (e) A representation of three independent dichlorofluorescin fluorometric assays, performed in quadruplicates, showing the kinetics of ROS generation in Huh8 cells (16 000 per well/96-well plate) after treatment with doses of H₂O₂, as indicated. The bottom part of the graph is enlarged and depicted below to show ROS generation in the lower range of H₂O₂. FL, Fluorescence units.