Y27362

To inhibit ROCK, cells were pre-treated for 20 min with Y27362 (Cat#Y0503, Sigma-Aldrich) at a final concentration of 10 μM. For suspension analysis, following detachment with Accutase (Cat#A1110501, ThermoFisher Scientific) for 5 min, cells were collected into complete DMEM (+/− Y27362), centrifuged at 500 xg for 5 min and resuspended in complete DMEM (+/− Y27362). To increase cell contractility, Calyculin A (Cat#C5552, Sigma-Aldrich) at final concentration of 25 nM was used and live imaging started immediately following its addition. Ezrin was inhibited with NSC668394 (Cat#341216, Merck Millipore) overnight at a final concentration of 10 μM or for 3 hr at 250 μM. For actin depolymerisation Latrunculin B (Cat#L5288, Sigma-Aldrich) was used at a final concentration of 20 nM. Equivalent volume of DMSO was used as controls. Accutase treatment and following incubation in suspension were performed at room temperature (22°C–25°C, RT). Accutase treatment had no effect on cell viability or ability to respread (Figure S1). Cells were also detached using EDTA (0.68M, in-house prepared solution).