Real-time PCR

Total cellular RNA was extracted from 16HBE and A549 using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA, using iScript cDNA Synthesis kit (Biorad, Hercules, CA, USA). Real-time quantitative PCR of IL-8 and IL-33 gene was carried out on Step One Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using specific FAM-labeled probe and primers (prevalidated TaqMan Gene expression assay for IL8, Hs00174103m1 and prevalidated TaqMan Gene expression assay for IL-33, Hs01125943m1; Assays on Demand, Applied Biosystems) as previously described [10, 19]. IL-8 and IL-33, gene expression was normalized to GAPDH endogenous control gene. Relative quantification of mRNA was carried out with comparative CT method (2^−ΔΔCT) and was plotted as fold-change compared to Non-treated (NT) sample, that were chosen as the reference sample.