FIG. 2.

Double-stranded RNA signaling pathways activate CXCL-8 transcription. (A) Expression of RIG-WT and constitutively active RIG-N proteins. Huh7 cells were transfected with plasmids expressing RIG-WT, RIG-N, or GFP, and whole-cell protein extracts were harvested 48 h later and analyzed by Western blotting with a polyclonal antiserum to RIG-I. Positions of the proteins are indicated with arrowheads. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cellular protein expression verified equal loading of proteins among the different conditions. (B) RIG-N activates CXCL-8 transcription. Huh7 cells were transfected with RIG-N- or GFP-expressing plasmids along with CXCL-8 reporter genes −1481-luc, −546-luc, or −162-luc, and luciferase readings were measured 24 h posttransfection. RLU, relative light units. Asterisks indicate significance of RIG-N luciferase values versus GFP values (*, P = 0.002; **, P = 0.017; ***, P = 0.003). (C) CXCL-8 promoter mutagenesis. Huh7 cells were transfected with RIG-N or GFP plasmids and the indicated CXCL-8 promoter constructs in the −162 backbone containing various mutations in transcription factor binding sites. Luciferase readings were measured 24 h posttransfection. Asterisks indicate that the luciferase values of mutant promoters compared to that of the wild-type −162 construct were significantly different (P, <0.01). (D) IPS-1 activates CXCL-8 transcription. Huh7 cells were transfected with IPS-1- or GFP-expressing plasmids along with wild-type −162 or mutant −162 promoters containing mutations in the NF-κB and ISRE binding sites, and 20 h posttransfection, cells were transfected with poly(I:C). Luciferase readings were measured 6 h later. The single asterisk indicates that IPS-1 luciferase readings were significantly different from the GFP readings (*, P <0.01). The double asterisk indicates that IPS-1-induced luciferase values from the ISRE mutant promoter were significantly lower than IPS-1 induced luciferase values from the wild-type promoter (**, P = 0.008).