5000 tension apparatus

Human aortic smooth muscle cells (HASMCs), purchased from ScienCell (U.S.A.), were cultured in SMC medium (ScienCell, U.S.A.) with 5% CO₂ at 37°C. Passages 4–7 cells were seeded (10⁵ cells/well) onto six-well Flexcell plates coated with collagen I, and when they reached 90% confluence, serum-free medium was added to induce quiescence for 24 h and replaced with fresh complete medium, then physiological stretch (10% elongation, 1 Hz) was applied by use of a computer-controlled Flexcell 5000-Tension apparatus (Flexercell Strain Unit, FlexCell International). The static control cells subjected to the same conditions as the experimental cells, except that they were not exposed to stretch. For inhibition of stretch-induced activation of transcription factors and signaling pathways, pharmacological inhibitors were added to the culture media 1 h before stretch treatment.

HASMCs were obtained from ScienCell and cultured in smooth muscle cell medium(SMCM) (USA) with 5% CO₂ at 37°C. Cells at passages 4–7 were used for experiments. HASMCs were seeded onto Flexcell 6-well plates coated with collagenI, when they reached 80∼90% confluence, serum-free SMCM was replaced to induce quiescence for 24 hr, then cells were exposed to cyclic stretch (16% elongation, 1 Hz) generated by a computer-controlled Flexcell 5000-tension apparatus for the indicated times. The apparatus was kept in a humidified incubator with 5% CO2 at 37°C. Cells cultured in static conditions were static controls. For inhibition of signaling pathway activation, specific Pharmacological inhibitors were added to SMCM 1 hr before stretch treatment.