Velocity statistics indicate robustness to substrate stiffness and sensitivity to cytoskeletal perturbation. (A) The velocity distribution of directed motion is independent of substrate stiffness. This provides further support, in addition to MSDs and VHCs, that substrate stiffness does not affect vesicle motion in fibroblasts. (B) The velocity autocorrelation function (VACF) quantifies persistence and indicates that vesicle motion is strongly perturbed by blebbistatin, which inactivates myosin-II. The strong negative peak (red squares) indicates vesicle motion is decreased when myosin-II is inactivated. Data shown for cells cultured on 10 kPa substrate.