Figure 3

Regulation of the IFN-β promoter by catenins and LEF1. (A - D) A549 or Vero cells were transfected with IFN-β luciferase gene reporter plasmid (indicated above the images) along with plasmids specified in column legends for 24 h and either left untreated or stimulated (by transfection) for 5 h with 500 ng of cellular RNA (isolated from uninfected A549 cells), viral RNA (isolated from PR8-infected A549 cells) or 1 μg of 5′-triphosphate modified RNA (pppRNA) per well. Luciferase activity of unstimulated or cellular RNA-stimulated and empty vector-transfected cells was taken as unity. (E) A549 cells were transfected with indicated plasmids for 24 h and conditioned media (donor cells) were harvested before the cells were lysed. The RIPA cell lysates were then analyzed for the efficacy of transfection by Western blotting (left panel). The conditioned media were applied to freshly plated A549 cells (acceptor cells) for 15 min and cells stimulated with conditioned media were then analyzed for phosphorylation of STAT1 at Y701 (right panel). Immunoblotting of β-actin served as loading controls. (F - H) Vero cells were transfected with luciferase reporter plasmids containing either the IRF3- (F), AP-1- (G) or NF-κB- (H) dependent binding region of the IFN-β enhanceosome along with plasmids indicated in column legends. The activity of reporter genes was determined 30 h after transfection. Luciferase activity of empty vector-transfected cells was always taken as unity. Mean values from three independent experiments are depicted in A, D, F, G and H, whereas B, C and E show representative images of three independent experiments.