Vesicle dynamics does not depend on substrate stiffness. (A) Mean squared displacement (MSD) of vesicle motion in untreated cells indicates no strong dependence on substrate stiffness. Vesicles in all cases show sub-diffusive behavior at shorter timescales transitioning to diffusive behavior at longer timescales. van Hove Correlation (VHC) functions for vesicles undergoing directed motion (B) and non-directed motion (C) show the full distribution of vesicle displacements is not sensitive to substrate stiffness. VHCs calculated for a timescale of 150 ms. Notice that VHCs of both directed and non-directed motion are strongly non-Gaussian, indicating cytoplasmic activity32. (Number of experiments/cells/trajectories - 10 kPa: 7/28/1211, 40 kPa: 7/24/3021, glass: 10/31/5605; S.E.M. smaller than symbol size).