FIG 3.

Antiviral effects of 3p10LG9 are RIG-I and IFNAR signal dependent. (A) Supernatant from U937-DC cells transfected with either immRNA or poly(I·C) was incubated on the HEK-293T cells that contain a luciferase reporter driven by an interferon-stimulated response element (ISRE-luc). Luminescence was measured at 6 h after incubation with the supernatant. Bars shown means ± SD for triplicate transfections, and data are representative of two independent experiments. Statistical significance was determined using a two-tailed Student t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). (B) U937-DC cells pretreated with either immRNA or poly(I·C) for 24 h were infected with DENV-2 TSV01 (MOI of 1). Infected viable cells were quantified using flow cytometry with antibodies targeting NS1 and the E-protein fusion loop (4G2) at 24 h after infection. Bars show means ± SD for triplicate transfections, and data are representative of two independent experiments. Statistical significance between the treatment methods within each cell type was determined using a two-tailed Student t test (*, P ≤ 0.05; ***, P ≤ 0.001). (C) Representative flow cytometry graphs of viable U937-DC cells stained with antibodies binding to NS1 and the E protein (4G2). (D) U937-DC-SIGN cells were transfected with immRNA or poly(I·C) or treated with IFN-β for 6 h before the addition of 10 μg/ml of either anti-IFNAR blocking antibody or an isotype control. Bars show means ± SD from transfections done in triplicate from two independent experiments. Statistical significance was determined using a two-tailed Student t test (*, P ≤ 0.05; ****, P ≤ 0.0001; ns, not significant). (E) Percentage of infection in U937-DC cells from panel D after infection with DENV-2 TSV01 (MOI of 1). Bars show means ± SD for transfections in triplicate from two independent experiments. Statistical significance was determined using a two-tailed Student t test (***, P ≤ 0.001; ns, not significant).