FIG. 6.

RIG-N stabilizes CXCL-8 mRNA. (A) Huh7 cells were transfected with RIG-N- or GFP-expressing plasmids, and 24 h later, cells were treated with 10 ng/ml TNF-α for 2 h to induce CXCL-8 mRNA. Actinomycin D was then added to cell cultures to stop transcription, and total cellular RNA was isolated at the indicated times. CXCL-8 mRNA was quantitated by real-time RT-PCR, as described in Materials and Methods. (B) Schematic representation of the CXCL-8 ARE reporter genes. The EGFP protein was placed under the control of a constitutively active CMV promoter. The control of mRNA stability was mediated by sequences in the 3′UTR. The dashed box indicates a 200-nucleotide stuffer sequence which lacks AREs in the vector construct or 237 nucleotides from the CXCL-8 3′UTR that contains AREs. Underlined are the ARE pentamers (AUUUA). (C) Cells (3 × 10⁴ cells per well) in black clear-bottomed 96-well plates were cotransfected with 25 ng of EGFP reporter vectors (CXCL-8 and WT) and 50 ng of modulator vectors (vector and RIG-N). After 48 h, the plates were read by bottom fluorescence. Data are presented as the means ± standard errors of four readings. ***, P values <0.001 using Student's t test.