Videos of LifeAct-GFP– or Fluo-4 Direct–stained MDA-MB-231 cells were analyzed by a custom MATLAB script. Cells were identified using image segmentation. Membrane protrusions in each branch channel at the time point when cells fully occluded the intersection were fitted by a second-order polynomial, and the prefactor (a) of the second order representing the membrane curvature was used. The original membrane curvature (ao) was obtained by the same method during cell migration inside the feeder channel and averaged over >10 consecutive time points. The percent change of membrane curvature was calculated by taking the absolute value of (a − ao)/ao.

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