Ad cmv null

Overexpression of human GATA6 gene was achieved using adenoviral constructs. Cells were transduced 24 h after seeding with 100 MOI of Ad-CMV-GATA6 (Vector Biolabs, #1027). Fresh media was added 96 h after virus transduction for additional 48 h. Ad-CMV-null (Vector Biolabs) was used as a control.

Cardiomyocytes were transduced with Ad-CMV-MKOS (Vector Biolabs) or Ad-CMV-Null (Vector Biolabs) at MOIs of 1–10 PFU/cell pre-diluted in cardiomyocyte maintenance media containing 2% FBS without BrdU. Unless otherwise noted in the figure or legend, cardiomyocytes were transduced with 5 PFU/cell. Cells were then maintained at 37°C for 24 hours before virus containing media was replaced with fresh cardiomyocyte maintenance media (without BrdU). Media was exchanged every other day thereafter. In a subset of experiments, as indicated in the text, media was replaced with ESC medium (Knockout DMEM/F12 (ThermoFisher, UK) supplemented with 15% Knockout Serum Replacement (ThermoFisher, UK), 1% StemPro non-essential amino acids (ThermoFisher, UK), 50 μM β-mercaptoethanol (Sigma-Aldrich, UK), 10 ng/ml of mouse leukaemia inhibitory factor (eBioscience, UK)) and was changed daily thereafter.

Deletion of the floxed Nfat5 exon was achieved by transduction with an adenovirus expressing Cre recombinase (iCre) under a cytomegalovirus (CMV) promoter (Ad-CMV-iCre, Cat. no. 1045, Vector Biolabs, Malvern, PA, USA). The virus was applied to the cells with a multiplicity of infection (MOI) of 500. An adenovirus with an empty CMV promoter (Ad-CMV-Null, Cat. no. 1300, Vector Biolabs) served as a control. Cells were utilized for experiments 48 h after exposure to adenoviral vectors. The knockout efficiency was controlled by determining the protein level of NFAT5 in cell lysates (Figure S2).

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.