Lyovec transfection reagent

HEK293T and HEK293T RIG‐I KO cells were grown in at 5% CO₂ and 37°C, in DMEM with 10% FBS in 6‐well plates to 60% confluence and cotransfected with firefly luciferase reporter plasmid (pLuc125 / 2.5 μg), Renilla luciferase reporter plasmid (pRL‐TK / 500 ng), and a plasmid carrying either the WT RIG‐I gene or mutant RIG‐I construct under the constitutively active CMV promoter (pcDNA 3.1 / 2 μg). The firefly luciferase gene is under the control of the interferon β promoter, and the Renilla luciferase plasmid is under the control of the constitutively active TK promoter. The plasmid transfections were carried out with X‐tremeGENE HP DNA Transfection Reagent (Roche). Cells were replated in 96‐well plates the next day at 2 × 10⁴ cells/well density and transfected with either 50 nM 5’ppp ds39 RNA or a titration of 700 nM, 70 nM, and 7 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen). After 20 h, the activities of firefly and Renilla luciferases were measured sequentially with the Dual‐Luciferase reporter assay (Promega). Data was collected in sextuplicate sets, and relative luciferase activities were calculated. Error bars represent the standard error of the mean (SEM). Protein expression was tested using western Blots with a primary α‐Myc antibody and β‐actin as a normalization control.

HEK293T cells were grown in 5% CO₂ and 37°C, in DMEM with 10% FBS in 6-well plates to 60% confluence and cotransfected with firefly luciferase reporter plasmid (pLuc125/2.5 μg), Renilla luciferase reporter plasmid (pRL-TK / 500 ng), and a plasmid carrying myc-tagged WT RIG-I gene under the constitutively active CMV promoter (pcDNA-3.1/2 μg). The firefly luciferase gene is under the control of the interferon β promoter, and the Renilla luciferase plasmid is under the control of the constitutively active TK promoter. The plasmid transfections were carried out with X-tremeGENE HP DNA Transfection Reagent (Roche). Cells were replated in 96-well plates the next day at 2 × 10⁴ cells/well density and transfected with indicated single-stranded and double-stranded RNAs (700 nM, or as indicated, in 110 μl volume) using Lyovec transfection reagent (InvivoGen). After 20 h, the activities of firefly and Renilla luciferases were measured sequentially with the Dual-Luciferase reporter assay (Promega). Each trial's number of replicates are shown in figure legends. Error bars represent the standard error of the mean (SEM).

HUVECs (70% confluence) were transfected with 0.1–1 ug/mL RIG-I or control agonist RNA (Cat: tlrl-hprna-100, Invivogen) according to manufacturers’ instructions. Specifically, The RIG-I or control agonist was diluted in Lyovec transfection reagent (Cat: lyec-12 Invivogen).

LyoVec transfection reagent and poly I:C (HMW) were purchased from InvivoGen (San Diego, CA). ELISA kit for IFN-λ1 was purchased from eBioscience Inc. (San Diego, CA). Rabbit antibodies against RIG-I and ISG15 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Rabbit antibody against TLR3 was purchased from Novus Biologicals (Littleton, CO). Rabbit antibodies against MxA and Actin were purchased from SIGMA-ALORICH (St. Louis, MO). Bafilomycin A1 was purchased from EMD Chemical, Inc (Gibbstown, NJ). siRNA against RIG-I and negative control siRNA were purchased from Qiagen (Cambridge, MA).

All culture plastic ware were obtained from Corning (Corning, NY, USA). Lyovec transfection reagent and Polyinosinic-polycytidylic acid (Poly I:C) (TLR3 ligand), Imiquimod (TLR7 ligand), ssRNA40 (TLR8 ligand), ODN2006 (TLR9 ligand) were purchased from InvivoGen (San Diego, CA, USA). All culture reagents were purchased from Gibco (Grand Island, NY, USA). Exosome-depleted fetal bovine serum (FBS) was purchased from System Biosciences, Inc. (Mountain View, CA, USA).