Tlrl pic

The synthetic lipopeptides Pam₃Cys-Ser-Lys₄ (P₃C) and fibroblast-stimulating lipopeptide 1 (FSL-1) were from EMC (Tübingen, Germany). LPS from Escherichia coli O111:B4, MDP, and poly(I:C) were purchased from Invivogen (San Diego, CA). Vaccigrade poly(I:C) (Invivogen, vac-pic) was used in most experiments. HMW (Invivogen, tlrl-pic) and LMW poly(I:C) (Invivogen, tlrl-picw) were used for comparison of high- and low-molecular-weight poly(I:C). Recombinant TNF was purchased from Peprotech (Rocky Hill, NJ).

Mice intraperitonally received 4 mg/kg polyinosinic-polycytidylic acid diluted in PBS (polyI:C, #tlrl-pic, invivogen). Control mice receive PBS and mice were harvested and tissue samples were analyzed at the indicated time points post treatment.

HEK293T cells were cotransfected with the IFN-β promoter–firefly luciferase (FLuc) reporter plasmid (pIFN-β-FLuc), p3×Flag-hHB, and the internal reference reporter TK-Renilla luciferase (RLuc) as an internal control (pRLuc-TK). The total amounts of the plasmid DNAs were equalized with the empty control vector p3×Flag-CMV-10 (p3×Flag-EV). At 24 hpt, the cells were infected with SeV or phosphate-buffered saline (PBS) for another 24 h. Then cells were lysed, and the activities of the reporter genes were determined using a dual-luciferase reporter assay system (10 pack; Promega). The luciferase induction mediated by IFN-β promoter (IFN-β-Luc) was presented as relative expression level of FLuc/RLuc. For the RIG-I- or MDA5-mediated response, HEK293T cells were cotransfected with pIFN-β-FLuc and pRLuc-TK as well as with pMyc-RIG-I, pMyc-MDA5, short poly(I·C) (catalog no. tlrl-picw; InvivoGen), or long poly(I·C) (catalog no. tlrl-pic; InvivoGen). The luciferase activities were measured at 24 hpt, and relative expression levels were calculated as described above.

HeLa and A549 cell lines were maintained in DMEM supplemented with 10% (v/v) foetal calf serum and 1% Penicillin/streptomycin at 37°C and 5% CO₂. Transfections of poly(I:C) (2 μg/ml, HMW, tlrl-pic; Invivogen) were performed using Lipofectamine 2000 according to the manufacturer's instructions. Four hours post-transfection, cells were collected for downstream applications. For IFN-β stimulation, HeLa cells were treated with 100U of recombinant human IFN-β (Peprotech, 300–02BC) for 4 h and harvested. Knock-down experiments were performed by two consecutive rounds of transfection with siRNA pools against ILF3 (L-012442–00-0005, Dharmacon) or EIF2AK2 (L-003527–00-0005, Dharmacon) in HeLa or A549s. As a negative control, a non-targeting siRNA pool was used (D-001810–10-05, Dharmacon). Individual siRNAs against both major ILF3 isoforms were adapted from Guan et al., (21 (link)). For NF110 (Sense-CUACGAGAGCAAAUUCAA C[dT][dT], antisense–GUUGAAUUUGCUCUCGUAG[dT][dT]), and NF90 (sense-G[mC]CCACC[mU]UUG[2flC]UU[2flU]UUAU[dT][dT], antisense- AUAA[mA]AAGCAAAGGUGG[2flG]C) siRNAs were used. Briefly, cells were seeded at 50–60% confluency and transfected with 25 nM siRNAs using Dharmafect. After 24 h, cells were split and transfected with a second round of siRNAs. Cells were collected for downstream processing 72 h after the first transfection.