2.12. VSMC transwell migration assay

Pretreated VSMCs were seeded into Transwell chambers with 8.0‐μm pores (BD Falcon, NY, USA) at a density of 2 × 10⁵ cells/well and cultured in serum‐free DMEM. DMEM supplemented with either Ang II (10⁻⁷ mol/L) or PBS was added to the bottom chamber. The VSMCs were allowed to migrate at 37°C in 5% CO₂ for 12 hours, after which the upper surface of the membrane was cleaned with a cotton swab. The migrated cells on the bottom surface of the membrane were fixed and stained with DAPI to visualize the nuclei. DAPI‐positive cells were counted in five random fields under a fluorescence microscope. Each experiment was conducted in triplicate.