Option monitor 3

TRIzol RNA Isolation Reagent (Life Technologies, Gaithersburg, MD, USA) was used for total RNA extraction from the lungs of mice. Total RNA quantity was determined using an absorption spectrometer (NanoDrop 2000 C, Thermo Fisher Scientific, Tokyo, Japan). The High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription. Real-time reverse transcription-PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) according to the instructions provided by the manufacturer. The levels of αSMA, collagen I, TGF-β, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase were quantified using commercially available kits (TaqMan Gene Expression Assays Mm00725412_s1, Mm00801666_g1, Mm01178820_m1, Mm00443258_m1 and Mm03302249_g1 respectively; Applied Biosystems). These primer sets were designed to span one intron to allow the identification of genomic contamination. The reaction protocol consisted of the following cycles: 95 °C for 15 min, 95 °C for 15 s, and 60 °C for 1 min for 50 cycles of PCR amplification on an Opticon 2 System (Bio-Rad). All data were analyzed on an Option monitor 3 (Bio-Rad).