2.5. A7r5 cell culture and siRNA transfection

A7r5 rat aortic SMCs (ATCC) were cultured in growth medium composed of Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% foetal bovine serum and 1% glutamine and maintained under humidified conditions at 37°C and 5% CO₂. Expression of NMII isoforms was inhibited by ON‐TARGETplus SMARTpool siRNAs specific for NMIIA and NMIIB, purchased from Horizon discovery. ON‐TARGETplus Non‐targeting Control siRNA was used as a control. Cells were seeded at a density of 1 × 10⁵ cells/mL and were transfected using DharmaFECT (Horizon Discovery), at a final concentration of 25 nmol/L in an antibiotic‐free Opti‐MEM medium according to the manufacturer's instructions. siRNA knockdown was analysed after 72 hours of transfection by Western blot using antibodies specific to NMIIA and NMIIB. Cells were lysed on ice, and samples were stored at −80°C.