Otoxicity of all extracts and eugenol was determined by MTT method on A549 and MDCK cells [43]. The concentration expected to reduce cell viability by 50 (IC50) was calculated. The maximal concentration with no cytotoxicity was employed because the optimal concentration.at 4uC for 1h, the fluorescence intensities (FI) have been measured at 610 nm soon after excitation at 587 nm utilizing a microplate reader (Tecan infinite M1000) and calculated as following:n 1XRFUsmp {RFUbackground n iDFIPlasmids ConstructionTo construct the BiFC plasmids, two segments from a red fluorescent protein (GenBank: HQ423140.1), corresponding to amino acids 1 to 159 and 160 to 262, respectively, were inserted into a pcDNA3.0 plasmid and named pMN and pMC, respectively. Human Bcl2 (NM_000633.2) were inserted into pMN and named pMN-Bcl2. Human Beclin1 (NM_003766.3) was inserted into pMC and named pMC-Beclin. To construct eukaryotic expression plasmid, human HMGB1 (NM_002128.4) and MyD88 (NM_001172567.1) were inserted into pcDNA3.0 and named pcDNA-HMGB1 and pcDNA-MyD88, respectively. To construct a pEGFP-LC3 plasmid, human LC3B (NM_022818.4) was inserted into a pEGFP-C1 plasmid. All constructs were verified by double enzyme digestion and DNA sequencing. Z’ 1{ 3scz z3sc{ Dmcz {mc{ DThe Z’-factor was a statistical parameter to quantify the suitability of a particular assay for use in a high-throughput screen [19]. The notations sc+ and sc- were the standard deviations of the negative control (NC) and blank group (BG), respectively, and mc+ and mc2 were the average values of the NC and BG groups, respectively. To observe the results of BiFC assay, A549 cells were seeded onto glass cover slip in a 12-well plate, and visualized under an upright fluorescence microscope (Nikon Eclipse 90i).Drug Screening Assay (BiFC Assay)A549 cells were seeded in 96-well plate for 24 h, after cotransfection with the corresponding plasmids for 6 h, IAV (MOI = 2.Desloratadine 0) and test drugs at the maximal no-cytotoxicity concentration were added, and incubated for 8 h, after treatmentPLOS ONE | www.plosone.orgPlaque Inhibition and Time-of-addition AssayPlaque inhibition assay was performed on MDCK cells [19]. Multiplicity of infection (MOI) was 0.001. The incubation time after absorption was 48 h. The supernatants were collected and the virus titers were determined by a plaque assay [19]. TheDrug Screening and Effect of Eugenol against IAVFigure 9. Eugenol inhibited the release of cytokines induced by IAV (A/ShanTou/169/06 (H1N1)). The treatments of untreated, virus only, ribavirin and Eug groups were same with those in Figure 6.Roxadustat The infection time was 24 h, MOI = 0.PMID:24732841 001. Data shown were the mean 6 SD of three independent experiments. *P,0.05, **P,0.01 vs the negative control group (virus only). doi:10.1371/journal.pone.0061026.gconcentration required to inhibit virus titer by 50 (EC50) was calculated. Antiviral index (AI) = IC50/EC50. Time-of-addition assay consisted of four tests [44]: 1) Before infection, the virus was pretreated by eugenol (5 mg/mL); 2) Before infection, the cells were pretreated by eugenol (5 mg/mL); 3) Eugenol was added during viral adsorption; 4) Eugenol (5 mg/mL) was added at different time points after virus challenge. MOI = 2.0. The incubation time after absorption was 12 h. The other performances were same with the plaque inhibition assay.as following, the concentration of 50 protection was defined as the EC50. Protection of test compound ( ) ODtest {ODNegative |100 ODMock {ODNegativeT.
