DsRNA and IFN treatments Cells were mock-treated, or treated with 8.5 nM poly I:C (GE Healthcare, Buckinghamshire, UK) in serum-free OptiMEM medium (Life Technologies) in the presence of 50 mg/ml DEAE- dextran (Pharmacia) for 1 hour. Full growth medium replaced OptiMEM for additional indicated amounts of time, unless otherwise noted. In all experiments, DEAE-dextran was used in controls not treated with poly I:C to ensure that subsequent cellular responses were not influenced by the former. It was previously determined that 8.5 nM poly I:C induces maximum protection in IRF32/292/2 MEFs [16]; as such, this concentra- tion was used to investigate the mediators of the observed antiviral response. For IFN treatments, cells were pretreated with 100 U/ml IFNb (provided by Dr. Brian Lichty, McMaster University) for 24 hours prior to challenge with HSVgfp. Supernatant transfer Supernatants from mock-treated cells and cells treated with 8.5 nM poly I:C for 5 hours were transferred to na¨ıve IRF32/ 292/2 MEFs and left on for another 5 hours. For experiments utilizing the iNOS inhibitor L-NIL, please refer to Inhibition of iNOS section, below. Cells were challenged with HSV-1gfp to determine if a soluble factor confers resistance to infection in the absence of both IRF3 and IRF9. Viral replication was quantified 24 hours post-infection as assessed by GFP fluorescence. To ensure no residual poly I:C in the transferred supernatants was conferring resistance to HSV-1 replication, absorbance of the supernatants was measured. In complete MEF media, poly I:C was serially diluted 1:5 with concentrations ranging from 2.261025–8.5 nM. This concentration range corresponds with that used in antiviral assays described previously [16]. A spectrophotometer was used to determine the absorbance of each concentration to derive a linear curve. To compare, absorbance of the supernatants at the time of transfer was also measured. Measurement of Nitric Oxide Production MEFs were seeded in 96-well plates to approximately 70% confluency. After 24 hours, the cells were incubated with poly I:C for 5 hours as described [16]. Following treatment with poly I:C, the concentration of nitric oxide in the supernatants of MEF cultures was assessed by measurement of NO22, an oxidized metabolite of nitric oxide. For this, a Griess reaction was performed as previously described [67]. Standards were prepared with known concentrations of NaNO2 (BDH) ranging from 0–20 mM prepared in alpha-MEM. Griess reagent (Sigma, USA) was prepared according to the manufacturer’s instructions and subsequently added to standards and samples. The absorbance of the supernatants in the plates was read at 550 nm after a 10 minute incubation at room temperature with Griess reagent. Nitrite Release WT and IRF32/292/2 MEFs were seeded at a confluency of ,80% overnight prior to treatments in 12-well plates for antiviral assays and in 96-well plates to assess nitrite release via Griess assay. The cells were treated with 0–200 mM DETA-NO (Sigma, USA) or with 0–200 mM of the control NONOate, DETA (diethylene- triamine; Sigma, USA) diluted in complete MEF media for 5 hours. Following treatment, cells were challenged with HSV-1 described earlier in an antiviral assay and viral replication was quantified as assessed by GFP fluorescence. Fresh DETA or DETA-NO was added after each medium change. dsRNA-Mediated Nitric Oxide Response Quantitative Real Time-PCR (qRT-PCR) RNA was isolated from cells using TRIzol reagent (Life Technologies). A random 6-mer primer (0.2 ng) and 50 U of Superscript II (Life Technologies) were used to reverse transcribe 300 ng of DNase-treated RNA (DNA-free kit, Ambion, Austin, TX) in a total reaction volume of 20 ml. Subsequently, qRT-PCR was performed in triplicate using Universal PCR Master Mix and gene-specific TaqMan primers (Life Technologies) in a total volume of 25 ml.Data were analyzed via the DDCt method. Gene expression was normalized to Gapdh, the housekeeping gene, and expressed as fold change over the mock-treated group (cells treated with DEAE-dextran alone). TaqMan specific primers used in this study include iNOS (Mm00440502_m1), IRF1 (Mm00515191_ m1) and Gapdh (Mm99999915_g1). Inhibition of iNOS The iNOS inhibitors AMG and L-NIL (Sigma, USA), were diluted in complete medium to a final in-well concentration of 10 mM. To investigate the efficacy of the iNOS inhibitors, cells were pretreated for 2 hours with iNOS inhibitors prior to a 5 hour treatment with poly I:C. RNA was then collected using TRIzol and prepared for qRT-PCR as described above. Expression of iNOS transcript levels both with and without inhibitor were compared using TaqMan specific primers to determine efficacy of each of the iNOS inhibitors used. To investigate the role of iNOS in the antiviral response, cells seeded the previous day in 12-well plates to 80% confluency were treated as described above. Cells were subsequently challenged with HSV-1gfp as indicated previously. NF-kB Inhibitor Preparation The NF-kB inhibitor Bay 11-7082 (EMD, Gibbstown, NJ) was prepared in 1% DMSO, and used at concentrations ranging from 1–10 mM. Cells were incubated with this compound for 0.5 hours prior to mock and poly I:C treatments. DMSO was included in mock- and poly I:C-treatments. Immunofluorescence Microscopy To determine the efficacy of Bay 11-7082 in inhibition of NF- kB in IRF32/292/2 MEFs, cells were seeded on glass coverslips overnight at 60% confluency. Cells were either mock- or poly I:C- treated with and without Bay 11-7082. Following 5 hours of treatment, cells were fixed with 10% formalin and subsequently permeabilized with 0.1% Triton X-100 diluted in 16 PBS for 10 minutes each. An overnight blocking step at 4uC followed in 16PBS with 2% goat serum. A 1:200 dilution of NF-kB p65 antibody (Santa Cruz Biotechnology, CA, USA) was added to the coverslips for 1 hour at room temperature. A secondary anti- rabbit IgG Alexafluor 488 antibody diluted 1:1000 (Life Technologies) was hybridized for 1 hour at room temperature. Nuclei were stained with Hoescht dye diluted 1:10000 for 10 minutes. All antibody and Hoescht dilutions were in 16PBS with 2% goat serum. Images were taken and analyzed using a Leica DM IRE2 inverted microscope with Openlab software (Improvision). Nuclear translocation of cells that received poly I:C and the inhibitor was plotted as a percent of untreated cells (mock- treated) corresponding to each dilution of Bay 11-7082. IRF1 siRNA An oligonucleotide specific for IRF1, (59-CAGACATCGAG- GAAGTGAAGGATCA-39) and a scrambled sequence (scr; 59- CAGTAGCGAAGGAGTAAGGACATCA-39) were designed (Thermo Scientific). The scrambled sequence was used to account PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31688