Transfection of Vero cells.

Vero cells grown in 10-cm dishes were trypsinized, and the number of cells was counted using a Countess II automated cell counter (Thermo Fisher Scientific). Next, 1 × 10⁶ cells were placed into each well of a 6-well plate and allowed to spread in an incubator (1.5 ml medium per well with 5% CO₂ at 37°C under humidified conditions) for 40 to 50 min before transfection. Transfection was performed using a mixture containing 4 μg of plasmids and 12 μl of the TransIT-293 transfection reagent (Mirus Bio LLC, Madison, WI). Briefly, the transfection reagent was mixed with 150 μl Opti-MEM reduced serum medium (Gibco) and incubated for 5 min at room temperature. This mixture was added dropwise to the plasmid DNA and then incubated for 20 to 25 min at room temperature before being added to the 6-well culture. At 24 h posttransfection (hpt), the culture supernatant was replaced with fresh DMEM supplemented with 10% FBS and antibiotics.