Quantification of Podosome Rosettes in HAECs and HMVECs

Following siRNA transfection, HAECs or HMVECs, were trypsinized and seeded on collagen I-coated coverslips in complete growth medium. Adherent cells were treated with VEGF-A₁₆₅ (25 ng/ml) and select experimental compounds [PKAi cocktail, PP2 (10 µmol/L), ML141 (10 µmol/L) or the appropriate vehicle] for a period of 24 hours, then fixed in 4% paraformaldehyde. To visualize actin and cortactin, the fixed cells were and stained with an anti-cortactin antibody (#05–180, clone 4F11; Millipore) and with TRITC-conjugated phalloidin. DAPI was also used to stain cellular nuclei. For high resolution images of podosomes and podosome rosettes, actin/cortactin-stained cells were imaged with a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Concord, ON, CA). For quantification of podosome-positive and podosome rosette-positive HAECs or HMVECs, images were acquired with a Zeiss Axiovert S100 microscope. Individual podosomes were identified by co-localization of F-actin and cortactin staining in a dot-like distribution (0.5–1 µm)^{59 (link)} while podosome rosettes were characterized by individual podosomes clustered into a ring or circular-like structure of dimensions (5–10 µm in diameter)^{59 (link)}. Identification of individual podosomes or podosome rosettes was quantified from at least three separate experiments in which 50–100 cells were examined per experiment.