mTORC2 inactivation reduces EC migration, matrix adhesion, and actin polymerization.

<p>HUVECs were transfected with siRNA targeting Akt1 (siAkt1), rictor (siRict¹) or raptor (siRapt). <b>A)</b> The ECs were seeded on gelatin-coated electrodes at equal density to reach confluence. The recovery of electric impedance was measured following delivery of a high electric current through an electrode to create a defect in the EC monolayer as described in Methods. Data are represented as the relative rate of migration per hour <i>versus</i> the control (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). HUVECs were transfected with siRict¹, or siAkt1, or treated with PP242. <b>B)</b> The EC were seeded equally on gelatin-coated electrodes, then adhesion was evaluated using electrical impedance measurements. Data are represented as the relative rate of adhesion <i>versus</i> the controls (n = 3 independent experiments, *<i>P</i><0.05 by ANOVA). <b>C)</b> The EC were serum-starved overnight, and stimulated with VEGF for 10 minutes or not (upper left). Fluorescence images of phalloidin-stained filamentous (F)-actin (green) and DNA (blue) are representative of 3 independent experiments illustrating a marked decrease in VEGF-stimulated F-actin among mTORC2-inactivated EC. <b>D)</b> HUVECs were transfected with siAkt1, or siRict¹, or control siNS. The EC were stimulated with VEGF, then globular (G)-actin and F-actin were separated as described in Methods. A representative Western blot illustrates the relative abundance of the F-actin and G-actin in ECs. <b>E)</b> Quantitation of the F-/G-actin ratio (n = 5 independent experiments, *<i>P</i><0.05 by ANOVA).