Acta2 hs00426835 g1

Total RNA was extracted from the human skin samples using the TRIZOL Lysis Reagent (Life Technologies, Carlsbad, Calif.) and RNeasy kit (Qiagen Inc., Valencia, Calif.). Superscript IV (Invitrogen) was used for reverse transcription. Gene messenger RNA (mRNA) expression levels were then assessed using quantitative real-time polymerase chain reaction (RT-qPCR) on a TaqMan Gene Expression Assays Step One Plus (Life Technologies), following the manufacturer’s protocol. TaqMan probes and premixed PCR primers for the human genes Collagen 1A1 (COL1A1: Hs00164004_m1), Fibronectin1 (FN1: Hs00365052_m1), actin alpha 2, smooth muscle (ACTA2: Hs00426835_g1), connective tissue growth factor (CTGF: Hs01026927_g1)‚ and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH: Hs02758991_g1) were obtained from Life Technologies. Gene expression levels were normalized to GAPDH and compared using the 2^-ΔCt method. Fold change (FC) in ΔΔCt in response to TGFβ was used to assess magnitude of the fibrotic response.

RNA was extracted using the RNeasy Kit (Qiagen), and complementary DNA (cDNA) was generated using the RNA-Ct Kit (Life Technologies). Gene expression was determined by quantitative polymerase chain reaction (qPCR) on ViiA 7 using Reverse Transcriptase qPCR Mastermix No ROX (RT-QPRT-032XNR, Eurogentec) and Assay-On-Demand premixed TaqMan probe master mixes [ACTA2Hs00426835_g1, Col1 Hs00164004_m1, ST-2 Hs00249384_m1, IL-33 Hs00369211_m1, ST2 Hs00545033_m1, TNFR1 Hs01042313-m1, TNFR2 Hs00961749-m1, and GAPDH (glyceraldehyde phosphate dehydrogenase) 4352665; Life Technologies]. The relative gene expression was calculated using the ΔΔCt method with the GAPDH gene for normalization of RNA levels.