F 12k medium

The A549 cell line (ATCC) was grown in media (F-12K medium with 10% [vol/vol] FBS [Atlanta Biologicals]) at 37 °C in 5% CO₂. Primary Human Bronchial Epithelial Cells (HBEpC; from ATCC) were cultured according to ATCC protocols. For the infection assay, cells were seeded at a density of 250,000 cells in a 35 mm glass-bottom dish and allowed to grow under standard culture conditions (37 °C, 5% CO₂); 24 h later the dish was washed with PBS and treated with A/Puerto Rico/8/1934 (H1N1) virus ((PR8, MOI = 2) in the infection medium (F-12K with 2% BSA fraction V [Thermo Fisher], 1% antibiotic-antimycotic [Thermo Fisher], and 1 μg/mL TPCK-treated trypsin [Worthington Biochemical]) for 1 h (37 °C, 5% CO₂). The cells were washed with the infection medium and incubated in the infection medium for 8, 6, or 24 h, as indicated prior to harvesting. Control cells were treated identically; however, no PR8 virus was added to the media.