Seakem le

A549 cells were treated either with NT siRNA, API5 siRNA, pLPC-Flag (control transfection), or pLPC-Flag-API5. At 24 h post transfections, cells were infected with X31 virus, a reassortant between PR8 and A/Aichi/68(H3N2) virus, at an MOI of 1. Supernatants collected after 24 h were analyzed for virus growth by plaque assay using MDCK cells. X31, being a low pathogenic virus as compared with PR8, forms clean plaques. MDCK cells were seeded in six-well plates (E106 cells per well) and the plates were incubated at 37°C overnight. Cell monolayers in all six-well plates were washed twice with DMEM and the culture supernatant containing virus was added in a volume of 200 μl at different dilutions. Each dilution was plated in duplicates. Plates were incubated with virus for 1 h followed by washing with DMEM with 0.3% BSA. The cells were overlaid with 1.6% Agarose (SeaKem LE, Cambrex, East Rutherford, NJ, USA) in L15 medium (2 × L15, 1M HEPES, 200 mM Glutamine, 50 mg/ml Gentamyin, NaHCO3, and penicillin streptomycin) with 1 mg/ml TPCK-treated trypsin (Sigma-Aldrich). The plates were incubated for 2–3 days, agarose was removed, cells were fixed with 70% ethanol for 5 min, and stained with crystal violet stain for 30 min. Cells were washed with distilled water, dried, and plaques were counted.

DNA was quantified with the Qubit fluorometer (ThermoFisher, USA), following the manufacturer's instructions. The integrity of the DNA (10 µL), with 2 µL of the 6X loading buffer (6X MassRuler, Loading Dye Solution, Fermentas) was evaluated after carrying out an agarose gel electrophoresis (SeaKem LE, Cambrex, USA) at 1%, dissolved in Tris-Acetate-EDTA Buffer (TAE), stained with GelRed (Gel Stain, Biotium, Cat: 41003). Electrophoresis was performed at 100 volts (FB1000 Power Source, Fisher Scientific, USA). To visualize the PCR amplifications and the RFLPs, electrophoresis was carried out on a 2% agarose gel, following the same methodology. The size of the amplified PCR, and of its fragments obtained after digestion with the enzymes (RFLP), was compared with a marker of 50 bp (DNA ladder GeneRuler, #SM0371, ThermoFisher, USA) or 100 bp (DNA ladder GeneRuler, #SM0241, ThermoFisher, USA). Image analysis was performed with a UV transilluminator (Slimline Series; Spectroline), the image was captured with an image digitizer (Enduro™ GDS, Labnet International, Inc., USA). Both the confirmation of the size of the amplified by PCR and the analysis of the RFLP were carried out with the TotalLab 1D software, version 14.0.