Fibroblast Response to TGF-β1 and Antifibrotic Treatments

RNAs were extracted from IPF and normal lung fibroblasts 24 h and 72 h after TGF-β1 treatment with either IFN-γ, PFD or both using RNeasy mini kits (Qiagen; Valencia, CA, USA) according to the manufacturer’s instructions. The RNAs were reverse transcribed into cDNAs, which were subjected to qPCR analyses using primer pairs specific to each of the following genes: COL1A1, ACTA2, MMP1, MMP3, MMP7, MMP8, MMP9, TIMP1, TIMP2 and YWHAZ (TaqMan™ Gene Expression Assay (FAM), Thermo-Fisher Scientific, Waltham, MA USA). qPCR was performed on an ABI Prism 7000 sequence detection system, and mRNA levels for each gene were analyzed with ABI Prism 7000 software (Applied Biosystems, Waltham, MA USA). Relative quantification of each target gene was performed using the comparative CT (2^-∆∆CT) method with YWHAZ (tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) used as a housekeeping gene for the normalization of mRNA expression levels as reported in previous studies [36 (link), 37 ]. Data were plotted as 2^-ΔΔCT (mean fold change) using Graph Pad prism software.