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. 2012 Jul 20;7(7):e41251. doi: 10.1371/journal.pone.0041251

Figure 6. ESEV PBM with intact CPSF30 binding site impairs NS1 inhibition of IFN-β promoter activation.

Figure 6

A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids, IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C) A549 cells were transfected with wt and mutant ESEA NS1 plasmid with intact CPSF30 site. After 48 hours, cells were lysed and analyzed for expression of NS1 in an immunoblots. The NS1 protein levels were normalized to corresponding β-actin level. Densitometry analysis was performed by ImageJ software.