Cryopreserved Human Coronary Artery SMC Culture

Cryopreserved human coronary artery SMCs were purchased from Thermo Scientific/Gibco (C-017-5C). They were cultured using either Human Vascular Smooth Muscle Cell Basal Medium (Life Technologies, M231500) with addition of growth supplement (SMGS, Life Technologies, S00725) and PEST (50U/50 μg/ml, Biochrom, A2212) or Smooth Muscle Cell Growth Medium (Sigma-Aldrich, #311–500) with addition of PEST in a standard cell culture incubator (37 °C, 95% air, and 5% CO₂). Smooth muscle cells from the human bladder were obtained in a prior study [40 (link)] and cryopreserved. After thawing, cells were cultivated in DMEM/Ham’s F-12 medium (Biochrom, FG4815) containing 10% fetal bovine serum (Biochrom, S0115) and PEST. All cells were used in passages 3–8. Adenoviruses were purchased from Vector Biolabs: Ad-h-MRTFA/eGFP (ADV-215499), Ad-h-MRTFB (ADV-215500), Ad-h-MYOCD (ADV-216227), Ad-CMV-Null (#1300), Ad-h-shSRF (shADV-224323), and Ad-GFP-U6-shRNA (#1122). Transductions were done at the indicated concentrations (multiplicities of infection, MOI). As negative controls for overexpression and knockdown we used Ad-CMV-Null and Ad-GFP-U6-shRNA, respectively. Transduced cells were harvested at 96 h, at144h, or at 192 h as indicated. The viral vector used for MYOCD encodes a transcript without exon 2a, expressed primarily in heart, but to some extent (≈ 25%), in SMCs.