FIG 5 .

IFN-β priming and ectopic expression of RIG-I change the pattern and the sensitivity of the IFN-β stimulation. (A and B) A549/pr(IFN-β). GFP reporter cells were treated or not with IFN-β for 6 h prior to transfection with 500 ng of various RNA/DNA hybrids (as indicated). Data are plotted as the percentage of GFP-positive cells measured by flow cytometry and are represented as means ± SD (n = 2). (Bottom panels) Intracellular level of RIG-I analyzed by Western blotting. As a loading control, the membrane was stained with Coomassie blue after immunoblotting. (C) A549 cells were transfected with an IFN-β-driven firefly luciferase reporter and a plasmid constitutively expressing Renilla luciferase. Additionally, cells were also transfected with a plasmid expressing RIG-I (RIG-I) or an empty vector (Ctrl.). After 24 h, the cells were transfected with 400 ng of various RNA or RNA/DNA hybrids as indicated. Luciferase activity was determined 20 h post-RNA transfection and normalized to Renilla luciferase activity and is reported as the fold increase compared to the level seen with the mock control without RNA. Data are represented as means ± SD (n = 2). The low transfection efficacy of A549 cells does not permit us to observe a significant increase in RIG-I expression by Western blot analysis (data not shown).