2.6. Influenza virus mini-genome assay

A549 cells were seeded at 3×10⁵ cells per well in 12-well plates and incubated overnight at 37 °C, with 5% CO₂ in a CO₂ cell culture incubator. The cells were transfected with pCDNA constructs of the influenza A/WSN/33 (H1N1) virus PB1, PB2, and PA (100 ng each) and NP (200 ng), the RNA polymerase II–driven Renilla luciferase reporter pRL-SV40 (Promega) (250 ng), and the influenza virus-specific RNA polymerase I–driven firefly luciferase reporter (vRNA Luc) (250 ng). The transfection was performed with TransIT-293 (Mirus) in OptiMEM (Invitrogen). Two hours after transfection, the media was supplemented with 1 mL of compound to their final concentrations. Twenty-four hours after incubation, cells were harvested, and firefly luciferase and Renilla luciferase expressions were determined using the Dual Luciferase Assay Kit (Promega).