Cc 3181

Human umbilical artery smooth muscle cells (UASMCs) (CC-2579, Lonza) were cultured on 0.05% Gelatin (from 2% Gelatin Solution in PBS, both from SIGMA) coated flasks in Smooth Muscle Cell Basal Media (SmBM, CC-3181, Lonza) supplemented with SmBM plus SingleQuots of Growth Supplements (CC-3182, Lonza) and maintained in a humidified incubator with 5% CO2 at 37 °C. Cells were passaged every other day at a ratio of 1:2. Cells used in this study were below passage 10. Human umbilical vein endothelial cells (HUVECs) were cultured on 0.5% Gelatin coated flasks in M199 medium (Gibco) supplemented with 10% fetal bovine serum and 1 ml EC supplement cocktail (contains 1ng/ml ECGF, 3 μg/ml EC growth supplement, 10unit/ml heparin, 1.25 μg/ml thymidine; all from Sigma-Aldrich). HEK 293 T (ATCC, CRL-11268) cells were cultured in DMEM media with 10% FBS in a humidified incubator with 5% CO2 at 37 °C. 293 T cells were passaged every other day at a ratio of 1:4.

Commercial primary human aortic smooth muscle cells (HAoSMCs) from one male donor with no reported cardiovascular comorbidities were obtained from Lonza (#CC-2571, Lot No. 0000369150, ascending aorta, Basel, Switzerland) and subjected to 0% or 12.5% substrate stretch as previously described [21 (link)]. In brief, custom made silicon chambers (inner dimensions: 4 cm² or 3 cm²) with replaceable silicon membrane bottoms were sterilized and coated with human fibronectin (5 μg/cm², #PHE0023, Life Technologies, Carlsbad, CA, USA). HAoSMCs at passage 6 were plated at sub-confluence (50,000 cells per cm²) and left to adhere overnight in basal growth medium (5% FBS, #CC-3181, Lonza, Basel, Switzerland). After 4 h of serum starvation in OptiMEM medium containing 0.1% FBS (#51985-026, Life Technologies, Carlsbad, CA, USA), the medium was changed to OptiMEM medium containing 1% FBS and 1.5 mM Ca, 1.5 mM PO₄. Controls were treated identically and supplement volumes replaced with PBS. Axial cyclic stretch was applied to the silicone chambers with a frequency of 70 cycles per minute. For comparison, HAoSMCs were also plated in fibronectin coated regular 6-well plates for expression analysis and chamber slides for immunofluorescence (IF) imaging, under identical medium conditions. After 8, 24 and 48 h medium supernatant, cell lysates or complete membranes were collected for further analysis.