Figure 1.

Induction of IFN-β promoter activity by the FMDV ncRNAs in HEK 293 cells expressing RLRs. (A) Cells (1 × 10⁶) were co-transfected with 50 ng of pIFNβ-FFLuc, 25 ng of pRL-TK, and increasing amounts of flag-RIG-I, flag-MDA5 or empty plasmid (1 and 10 ng). A constitutively active RIG-I, CARD2 (2 ng) served as positive control. After 24 h, cells were mock-treated or stimulated with 0.3 μg (0.2 μg/mL) or 3 μg (2 μg/mL) of the indicated ncRNA. Cells were harvested 24 h later and dual-luciferase assay was performed. The values represent the relative firefly luciferase activity normalized to Renilla luciferase activity and expressed as fold differences relative to mock-transfected cells. Data are representative of two independent experiments and error bars indicate mean +/− SD; (B) Expression of RLRs in transfected 293 cells. HEK293 cells were transfected with 100 ng of flag-RIG-I or flag- MDA5. Forty-eight hours later, cell lysates were prepared and analyzed by immunoblot with anti-flag, anti-RIG-I, anti-MDA5 or anti-β-tubulin antibodies.