Fig 1: Extracellular matrix (ECM) gene expression in primary human cardiac fibroblasts and primary human coronary artery vascular smooth muscle cells varies in different in vitro phenotypes. (A) Schematic overview of cell types used and experimental set-up. Commercially available human primary left ventricular cardiac fibroblasts (CF, Cat# CC-2904, Lonza, Basel, Switzerland) and primary human coronary artery vascular smooth muscle cells (VSMC, Cat# 121 0612, Provitro, Berlin, Germany) from three organ donors for each cell type were seeded in 6-well plates in passage six. CFs were cultured in growth media containing 10% fetal bovine serum (FBS, Cat# 16140071, Thermo Fischer Scientific, Waltham, MA, USA) and stimulated with 10 µmol/l transforming growth factor (TGF) ß receptor (TGFßR) I inhibitor (Cat# SB421542, Sigma, St. Louis, MI, USA) and 25 µmol/L Rho-associated kinase (ROCK) inhibitor (Cat# Y27632, BD Biosciences, Heidelberg, Germany) for 48 h, or 10 ng/mL TGF-ß1 (Cat# 7754-BH, R&D Systems, Minneapolis, MN, USA) for 24 h. VSMC were cultured in growth media containing 10% FBS or in serum starvation media containing 0.5% FBS for 72 h, with subsequent stimulation with 10 ng/mL TGF-ß1 for 24 h. For all experiments, three technical replicates were used for RNA sequencing, which was performed in parallel for all samples to minimize technical variation. RNA was isolated with Qiagen Mini Plus Kit (Cat# 74136, Qiagen, Hilden, Germany) and quality checked with a bioanalyzer (Cat# 5067-1511, Agilent RNA 6000 Nano Kit, Agilent Technologies, Santa Clara, CA, USA) before sending to Eurofins for RNA sequencing. (B) Heatmap of row-z scores across expression values for ECM genes after the different treatments. STAR (2.7.3) [57] was used to map samples to the GRCh38 reference genome, and mapped reads were passed to Salmon (1.2.0) [58] for transcript-level quantification on the TPM level.