Cell Culture
BEAS-2B cells and primary normal human bronchial epithelial cells (NHBE cells) were purchased from ATCC (Manassas, VA, United States) and Lonza (Tokyo, Japan), respectively, and were cultured according to manufacturer recommendations. BEAS-2B cells are virus-transformed human bronchial epithelial cells. Briefly, BEAS-2B cells (2.0 × 105 cells/well) and NHBE (1.0 × 104cells/well) cells were cultured with serum-free Bronchial Epithelial Growth Media (BEGMTM, Lonza) to a confluence of 80-100%, which usually took 3 days. Cells were afterwards stimulated with cytokines and/or TLR ligands for 24 hours in a 24-well culture plate unless otherwise specified. For inhibitory assays, BEAS-2B cells were co-incubated with inhibitors for 2 hours prior to cytokine stimulation. To evaluate the activity of inhibitors, percent (%) inhibition at each concentration of the inhibitor was calculated using the following equation:
% inhibition = (1-A/B) x 100,
where A and B were culture-media CCL5 concentrations of BEAS-2B cells grown with poly (I:C) after pre-incubation treatment with an inhibitor and a vehicle, respectively. We then performed a curve fitting analysis using the following equation:
Y = Bottom + X * (Top - Bottom) / (EC50 + X)
and calculated the maximal inhibitory effect of inhibitors.