Expression of ISG's was assessed with real-time quantitative PCR assay for the corresponding mRNA level. For these experiments, U3A and U3A-STAT1 cells were first treated with the programmed combination of compound and IFN-b for 12 h and then were washed twice with cold Dulbecco's PBS followed by lysis with Cells-to-cDNA II lysis buffer (Life Technologies) and treatment with DNase according to the manufacturer's instructions. The U3A cells were obtained from G. Stark (Cleveland Clinic) and complemented with STAT1 to generate U3A-STAT1 cells as described previously [13] . A 25-ml aliquot of the cell lysate was used to generate cDNA using the High Capacity Reverse Transcription Kit (Life Technologies). The resulting cDNA was quantified using the Quant-iT OliGreen ssDNA kit (Life Technologies). Average cDNA concentration was 71625 ng/ml. Subsequent PCR assays were performed in adherence with MIQE guidelines [60, 61] , including the design of assays for ISGs (OAS1 and GBP1) and normalizer gene ornithine decarboxylase antizyme (OAZ1). The normalizer gene OAZ1 was selected and validated for cell samples treated with and without IFN-b. In brief, candidate normalizer genes were selected from a combination of invariant genes selected from previous microarray data [11, 62, 63] and prior large-scale analyses of publicly available microarray data [64, 65] . These candidates were then tested using real-time quantitative PCR assays. Comparison of candidate normalizer gene expression between various IFN treatment and infection conditions using multiple software packages [66 68] led to the selection of the OAZ1 as the normalizer gene. Primers and probes for real-time quantitative PCR assays were designed using the ProbeFinder design algorithm (Roche Applied Science). For OAS1, 59ggtggagttcgatgtgctg-39 and 59-aggtttatagccgccagtca-39 were used as forward and reverse primers, along with UPL probe #37 (Roche Applied Science). A plasmid containing OAS1 transcript variant 2 cDNA (Ref ID NM_002534, Origene, Rockville, MD) was used as a standard for absolute quantitation of OAS1 copy number. For GBP1, 59-ttccaaaactaaaactctttcagga-39 and 59tgctgatggcattgacgtag-39 were used as forward and reverse primers, along with UPL probe #85. A plasmid containing the GBP1 cDNA (Clone ID: 3606865, Thermo Open Biosystems, Huntsville, AL) was used as a standard. The IDT PrimeTime pre-designed assay Hs.PT.42.328511.g (Integrated DNA Technologies, Coralville, IA) was used for OAZ1. A cDNA vector was used for OAZ1 as well (Clone ID: LIFESEQ913650, Thermo Open Biosystems). Data were collected on a LightCycler 480 instrument (Roche Applied Science). Quantification cycle values were calculated using a second derivative maxima algorithm as implemented in the Lightcycler 480 software.