sIL-6R upregulates IL-32 expression during IAV infection. (a) A549 cells were incubated with recombinant human IL-6Rα (40 ng/ml) for the indicated times, and the level of IL-32 mRNA was examined by real-time RT-PCR. (b) A549 cells were incubated with IL-6Rα at the indicated doses, and the level of IL-32 protein was examined by western blotting. (c) MRC-5 cells were incubated with IL-6Rα at the indicated doses, and the level of IL-32 mRNA was examined by real-time RT-PCR. (d and e) A549 cells were transfected with pCMV-sIL-6R for the indicated times; the IL-32 mRNA levels were determined by real-time RT-PCR (d), and the IL-32 protein was assessed by western blotting (e). (f and g) A549 cells were transfected with shRNA-sIL-6R or shRNA-control for 24 h (f) or 48 h (g) and infected with IAV (MOI=1) for 6 h. The levels of IL-32 mRNA (f) were quantitated by real-time RT-PCR, and western blot analysis was performed to assess the IL-32 levels in the cell lysates (g, upper panel). IAV NP mRNA was detected by semiquantitative RT-PCR (g, lower panel). Data shown are mean±s.e.; n=3. *P<0.05; ns, not significant. IAV, influenza A virus; MOI, multiplicity of infection; sIL-6R, soluble interleukin-6 receptor.