Probing roles for tension in VL initiation. (a) MVECs were pretreated with Y27632, ML-7, blebbistatin, or vehicle (control) for 30 min and then subjected to probe micro-wounding in the presence of drug. Velocity of wound retraction (i), time until first appearance of VL (ii), and the percentage of pores recovered by 5 min after wounding (iii) were measured. n > 15. (b) Schematic of a uniaxial de-stretch/compression experiment (i). MVEC monolayers expressing actin-GFP and mYFP were grown on 20% prestretched silicone membranes and imaged during an acute 10% de-stretch. An inter-endothelial junction region is shown as either overlay of the stretched (magenta) and 90 s after de-stretch (green) time points (ii) or as sequential time series (iii). Cyan arrows highlight putative VL formed after de-stretch. See Video 8. (c) Analysis of de-stretch–induced VL formation at ends versus sides of cells (Katsumi et al., 2002). n = 11. All values are mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05. Bars, 5 µm.