Poly 1 c lmw

A cannula was inserted in trachea from adult or neonatal mice and repeated BAL were made with PBS. AMs were isolated after centrifugations (≈2 × 10⁴ AMs in BAL from 1 neonate versus ≈1 × 10⁵ AMs in BAL from 1 adult mouse) and 1 × 10⁵ AMs were plated in 96-well cell culture plates in RPMI supplemented with L-glutamine 2 mM, FCS 5%, and antibiotics for 24 h to allow for adhesion. AMs were then exposed to recombinant RSV expressing mCherry^{30 (link)} or not (WT rRSV) or UV-inactivated mCherry-RSV (the same batch exposed 20 min to UV) at multiplicity of infection (MOI) = 5 or Hep2 cell culture supernatant (Mock), or stimulated with imiquimod 100 µg/mL (Invivogen), CpG-B 100 µg/mL (Sigma-Aldrich), Poly(I:C) LMW 100 µg/mL (Invivogen), LPS 10 µg/mL (E. coli 0111:B4, Sigma-Aldrich) or Poly(I:C)-LMW/LyoVec 0.1 or 1 µg/mL (Invivogen). In some experiment, adult C57BL/6 AMs were infected in the presence of anti-IFNAR1 antibody (BioXcell, MAR1-5A3, 5 or 10 µg/mL) or control isotype (MOPC-21). After 24 h, supernatants were collected and cells were frozen in NucleoSpin^®RNA XS Kit (Macherey-Nagel) lysis buffer for qRT-PCR analysis. Cell preparations from WT or deficient mice (pups or adults) were performed simultaneously. However, the analyzes were separated for a better understanding of results.

Information about plasmids expressing each gene is as follows: IRF3(5D), RIG-I, MAVS [33 (link)]; TLR3 (a kind gift from Dr. Kui Li at the University of Tennessee). The antisense oligonucleotides against nc886 and non-target control were described in [11 (link)]. “anti-nc886” and “anti-control” in the text and figures indicate “anti886 75_56” and “anti_vt 21_2”, respectively, in the reference. siRNAs directed against PKR and control were Stealth RNAi™ siRNA (Invitrogen), as described in [34 (link)]. Poly(I:C)-HMW(1.5–8 kb) and Poly(I:C)-LMW (0.2–1 kb) were purchased from Invivogen (San Diego, CA, USA). In most experiments, Poly(I:C)-LMW was used unless otherwise mentioned in figure legends. Plasmids and Poly(I:C) were transfected with Lipofectamine™ 2000 reagent (Invitrogen) per the manufacturer’s instructions. Anti-oligos and siRNAs were with Lipofectamine^TM RNAiMAX reagent (Invitrogen).

Trizol (Takara Bio), SYBR Green (Bio-Rad), RNase I (Ambion), Flag antibody-conjugated beads (Bimake), dual-specific luciferase assay kit (Promega), polybrene (Millipore), poly(I:C)-HMW (Invivogen), poly(I:C)-LMW (Invivogen), 5’ppp-dsRNA (Invivogen), DNAase I, 3 × Flag peptide (Sigma), Z-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), first-strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), and ELISA kits for murine IFN-β and TNF (BioLegend); mouse monoclonal antibodies for Flag and β-actin (Sigma), HA (Origene), p-IRF3, p65 and p-p65 (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), p-TBK1 and TBK1 (Abcam), and ZFYVE1 (ABclonal); Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen); and HEK293 (American Type Culture Collection) and THP1 cells (American type culture collection) were purchased from the indicated companies. HFFs were provided by Dr. Min-Hua Luo (Wuhan Institute of Virology, CAS). SeV (Cantell strain), EMCV (BJC3 Strain) and VSV (Indiana Strain) were previously described [28 (link), 30 (link)].