FIGURE 3.

IAV induces IL-35 expression at the transcriptional level. A, A549 cells were transfected with luciferase reporter plasmids containing the IL-35/EBI3 (EBI3-Luc) or IL-35/p35 promoter (p35-Luc) along with pRL-TK and either infected or not infected with IAV (m.o.i. = 1) for 24 h. Luciferase activity was measured after 12 h-serum starvation. B, A549 cells were transfected with EBI3-Luc or a series of truncated and mutated plasmids containing the IL-35/EBI3 promoter along with pRL-TK; cells were then infected with IAV (m.o.i. = 1) for 24 h. Luciferase activity was measured as in A. C, experiments performed as in B, except p35-Luc or a series of truncated and mutated plasmids containing the IL-35/p35 promoter. D, A549 cells were co-transfected pCMV-p50 or pCMV-p65 with EBI3-Luc or p35-Luc along with pRL-TK; cells were then infected with IAV (m.o.i. = 1) for 24 h. Luciferase activity was measured as in A, and the expression of p50 and p65 was detected by Western blot (lower panel). E, experiments were performed as in D, except where shRNA-p50 or shRNA-p65 were used. The silencing efficiencies for p50 and p65 were measured by Western blot (lower panel). F and G, experiments were performed as in D and E, except pCMV-CREB (F) or shRNA-CREB (G) were used. CREB expression was detected by Western blot (lower panel). n.s., not significant. H and I, A549 cells were infected with IAV (m.o.i. =1) for 12 h. ChIP assays were performed using a NF-κB/p65 antibody (Ab, normal IgG as control). Bond DNA fractions on the IL-35/EBI3 (H) or IL-35/p35 promoter (I) were measured using qRT-PCR utilizing the indicated primers. Bar graphs represent the mean ± S.D. n = 3. *, p < 0.05.