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. 2011 Jan 13;286(12):10568–10580. doi: 10.1074/jbc.M110.206896

FIGURE 3.

FIGURE 3.

ARL16 siRNA results in an enhanced antiviral response. A, effects of ARL16 RNAi plasmids on the expression of transfected ARL16. 293T cells (2 × 105) were transfected with expression plasmids for Flag-ARL16, with Flag-MITA as control (0.5 μg each), and the indicated RNAi plasmids (1 μg). At 48 h after transfection, cell lysates were analyzed by Western blot with anti-Flag and anti-GAPDH antibodies. B and C, ARL16 RNAi plasmid potentiates RIG-I, but not RIG-I-NT and VISA-, TBK1-, and TRIF-induced activation of IFN-β promoter. 293T cells (2 × 105) were transfected with the indicated reporter plasmid (50 ng), pRL-TK Renilla luciferase plasmid (50 ng), and the indicated ARL16 RNAi (1 μg). At 40 h after transfection, luciferase assays were performed. D, ARL16 RNAi enhances RIG-I, but not and VISA-, TBK1-, and TRIF-mediated gene expression. 293T cells (2 × 105) were transfected with indicated plasmids. Twenty-four hours after transfection, total RNA was isolated and RT-PCR was performed using indicated primers. E, ARL16 RNAi plasmid potentiates SV-induced, but not TLR3-mediated activation of IFN-β promoter. Experiments were carried out as in A and B. For polyI:C treatment, cells were also transfected with expression plasmids for TLR3 (100 ng). At 40 h after transfection, cells were infected with SV, treated with polyI:C (2 μg/ml) or left untreated for 12 h before reporter assay. F, ARL16 RNAi enhances SV-induced, but not TLR3-mediated gene expression. The transfection and treatment were carried out as in E, total RNA was isolated, and RT-PCR was performed using indicated primers. G, ARL16 RNAi enhances NDV-eGFP replication. 293T cells (2 × 105) were transfected with the indicated plasmids. At 20 h after transfection, cells were infected with NDV-eGFP at MOI 0.001. At 40 h after infection, virus replication was determined by GFP expression visualized by fluorescence microscopy. The GFP-positive cells were counted, and the percentage was calculated. H, knockdown of ARL16 potentiates the RIG-I-mediated anti-VSV response. 293T cells (2 × 105) were transfected with indicated plasmids (0.5 μg each). At 30 h after transfection, cells were infected with VSV (MOI = 0.01), and supernatants were harvested at 12, 18, 24, and 30 h post-infection. Supernatants were analyzed for VSV production using standard plaque assays. Plaques were counted and titers calculated as plaque-forming units (pfu/ml).