Mdck siat1 cells

To propagate influenza viruses, MDCK-SIAT1 cells (Millipore Sigma) were used. Cells were maintained with complete media comprising Dulbecco’s modified Eagle’s medium high glucose (DMEM; ThermoFisher) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gemini Bio-Products), 100 units ml⁻¹ penicillin (ThermoFisher), 100 μg ml⁻¹ streptomycin (ThermoFisher), and geneticin (1 mg ml⁻¹) (ThermoFisher). To develop constitutively PB1- or HA-expressing MDCK-SIAT1 cells, one plasmid encoding both puromycin resistance and influenza genes was transfected into MDCK-SIAT1 cells using Lipofectamine 2000 (ThermoFisher). Two days post transfection, cells were transferred from 6-well plates into 10-cm dishes containing DMEM media with 10% bovine serum (Gemini Bio-Products), penicillin, streptomycin, geneticin, and puromycin (0.25 μg ml⁻¹; ThermoFisher) for selection. Medium was changed every 48 h. Clonal selection was performed using 8 or 10 mm cloning cylinders (Fisher Scientific) about 2 weeks after the transfection. Clonal cell lines were screened using a reporter virus prepared on a polyclonal cell line. To rescue influenza viruses, Flp-In 293 cells (ThermoFisher) were transfected as described below.