Study of magnitude of stretch at 5%. TM expression on HUVECs is dependent on the magnitude of stretch. (A) Confocal images of HUVECs stretched for 24 hours at 5% on a flat membrane: DAPI in blue, F-actin in red, TM in magenta, and VWF in green on the same sample. Objective, original magnification ×40; scale bar, 20 μm. The arrow represents the direction of the uniaxial cyclic stretch. Z-stack sum projection. (B) Frequency distribution of TM and VWF expression (intensity of fluorescence I [AU]) for 1 representative experiment. Unstretched, light blue; stretched for 24 hours at 10%, orange; and stretched for 24 hours at 5%, green. Insets: relative protein expression fold change (bar graph), mean ± SD values of unstretched and stretched for 24 hours at 10% vs stretched for 24 hours at 5%, N = 2 independent experiments, respectively (medium and multiwell Curibio cytostretcher membranes were used to facilitate averaging across duplicates). ∗∗∗∗P < .0001. Every black dot represents 1 independent experiment.