Promofectin

Rat aortic valve interstitial cells (AVICs) were collected from aortic valve of 10-week-old Sprague Dawley females. AVICs were isolated as previously described by Gould et al., 2010 [30 (link)]. Briefly, valves leaflets were isolated and submitted to collagenase type II digestion. To isolate AVIC, leaflets were cut into small pieces and placed into collagenase solution during 8 h at 37 °C. After isolation, AVICs were cultured in DMEM-GlutamaX (Invitrogen, Thermo Fischer Scientific, Carlsbad, CA, USA) supplemented with 10% FCS (Invitrogen). AVICs were used between passage 3 and 7 for this study. AVIC were seeded into 6-well plates at 200,000 cells of density and 500 ng of CMV-Krox20 or CMV-GFP were transfected using Promofectin (Promocell, Heidelberg, Germany). Cells were lysed 24 h, 48 h, or 72 h after transfection, and RNA was isolated using NucleoSpin RNA/Protein kit (Macherey-Nagel, Düren, Germany).

HEK 293 cells were cultivated to 70–80% confluency before transfection. For transfection of Fgf8 and Hsc70 cells were seeded at 2×10⁶ cells in a 10cm plate. After 24 hours cells were transfected according to the manufacturer’s protocol using Promofectin (Promocell). Briefly two mixtures containing DNA/serum free DMEM and Promofectin/serum-free DMEM were combined incubated for 20 min at RT and added to the cells. 24 hrs after transfection the culture medium was replaced and Bafilomycin A1 was added. 24 hrs later the cells were lysed in sodium dodecyl sulfate (SDS)–sample buffer containing 100 mM dithiothreitol (DTT) and subjected to Western blot analysis. Activated Erk was monitored using an antibody against phosphorylated Erk (phospho-p44/42, Cell Signaling). For the loading control the membrane was stripped (62.5 mM Tris, pH 6.8, 2% SDS, 0.8% DTT) and reprobed with an Erk antibody (Erk 1 (K-23) Santa Cruz). Blots were stained using the enhanced chemiluminescence system (Thermo Fisher Scientific). The quantification of proteins bands in Western blot analysis was performed using the program ImageJ.

Plasmids encoding Perceval, PercevalHR and pHRed were obtained from Addgene (Cambridge, MA). HCASMCs expressing PercevalHR or pHRed were transiently transfected using either FuGENE6 (Promega UK, Southampton, UK) or Promofectin (Promocell, Heidelberg, Germany) following manufacturer’s instruction. HCASMCs were seeded 24 h prior to transfection at a density of 1.25 × 10⁵ per well in 35 mm glass-bottom dishes (Greiner Bio-One, Gloucestershire, UK). The cells expressing Perceval and PercevalHR were used after 24–48 h of transfection, and cells expressing pHRed were also used after 24-48 h of transfection. The cells expressing PercevalHR based on a conventional 3rd generation lentivirus packaging system which has been described previously were used in PercevalHR in vitro calibration experiments (Yang et al., 2017 (link)).