Fig. 3.

Both MDA5 and RIG-I are involved in cytoplasmic poly(I:C)- and SeV-triggered signaling. (A) Effects of MDA5 RNAi plasmids on the expression of endogenous MDA5. The 293 cells (1 × 10⁶) were transfected with a control or MDA5 RNAi plasmids (10 μg of each). Forty-eight hours after transfection, cell lysates were analyzed by Western blots with anti-MDA5 (Upper) or anti-GAPDH (Lower) antibody. (B) Effects of RIG-I RNAi plasmids on the expression of RIG-I. The 293 cells (5 × 10⁵) were transfected with HA-tagged RIG-I and IKKε plasmid (1 μg) and a control or RIG-I RNAi plasmids (2 μg of each). Forty-eight hours after transfection, cell lysates were analyzed by Western blot with anti-HA antibody. (C) Effects of MDA5 and RIG-I RNAi plasmids on cytoplasmic poly(I:C)-triggered ISRE activation. The 293 cells (1 × 10⁵) were transfected with ISRE luciferase reporter plasmid (0.05 μg), control, or MDA5 or RIG-I RNAi plasmids (0.5 μg of each) as indicated. Thirty-six hours after transfection, cells were further transfected with poly(I:C) (4 μg, gray bars) or buffer (white bars) by Lipofectamine for 12 h before luciferase assays were performed. (D) Effects of MDA5 RNAi plasmids on SeV-triggered activation of ISRE and the IFN-β promoter. The 293 cells (1 × 10⁵) were transfected with ISRE or the IFN-β promoter luciferase reporter plasmid (0.05 μg) as indicated and MDA5 RNAi plasmids (0.5 μg of each). Twenty-four hours after transfection, cells were infected with SeV (gray bars) or left untreated (white bars) for 12 h before luciferase assays were performed. (E) Effects of RIG-I RNAi plasmids on SeV-triggered activation of ISRE and the IFN-β promoter. The experiments were performed as in D. (F) Effects of a combination of MDA5 and RIG-I RNAi plasmids on SeV-triggered activation of ISRE and the IFN-β promoter. The experiments were performed as in D. The 293 cells were transfected with 0.5 μg of MDA5 or RIG-I RNAi plasmid alone, or a combination of 0.25 μg of each. Relat. Lucif. Act., relative luciferase activity.