Live AFM Imaging of VSMC Topography

Contact mode live AFM imaging (MFP-3D-BIO, Asylum Research, Santa Barbara, CA) was used to study live VSMC topography. A 30 × 30 μm area of the cell surface was scanned at a digital density of 512 × 512 pixels using a stylus AFM probe (model MLCT-C, k = 15 pN/nm, Bruker, Santa Barbara, CA). The cell surface scanning frequency was 0.3 Hz. All experiments were conducted in the CO₂-independent medium at room temperature for a period of 1–2 h.^{28 (link),31 (link)} AFM height images were used to compute actin stress fiber density (Figure S2). AFM deflection images were used to compute VSMC cortical actin stress fiber orientation using a modified method of Karlon et al. (Figure S3).^{33 (link)}