Figure 3. Apoptosis as well as IFN-β induction by pppRNA and poly(I:C) are mediated via IPS-1 in melanoma, but apoptosis is independent of IFN-β secretion.

(A) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) 48 hours after transfection of siRNAs specific for RIG-I, MDA-5, or IPS-1 or control siRNA. IFN-β expression was analyzed by quantitative RT-PCR. (B) 1205Lu cells were treated with pppRNA or poly(I:C) (5 ng/ml) 48 hours after transfection of siRNAs specific for IPS-1 or control siRNA, and rates of apoptosis were determined by FACS. Annexin V–positive and propidium iodide–negative cells are represented. *P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA1ds. (C) 1205Lu cells were transfected with vectors expressing wild-type NS3-4A (NS3/4) or an inactive mutant form (mNS3/4) (51). Twenty-four hours after vector transfection, cells were treated with pppRNA. Left: Apoptotic and dead cells (AN⁺/PI⁺) were measured. *P ≤ 0.05 compared with mNS3/4- or mock-transfected cells treated with pppRNA. Right: Analysis of IFN-β expression by quantitative RT-PCR. (D) 1205Lu cells were treated with siRNA specific for the type I IFN receptor (IFNAR) or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A. Left: quantification of IFNAR mRNA. Middle: Quantification of IFN-β expression. Right: FACS analysis of apoptotic cells (AN⁺/PI^–) treated with pppRNA, poly(I:C), or transfection reagent alone. (E) 1205Lu cells were treated with an IRF-3–specific siRNA or control siRNA and pppRNA as described for A. Left: Analysis of IRF-3 mRNA expression. Right: Analysis of apoptotic cells (AN⁺/PI^–). For all panels, mean ± SD of 3 independent experiments is shown.