Videos of MDA-MB-231 LifeAct-Ruby2 or MIIA-GFP cells were analyzed by a custom MATLAB script. Cortical actin and MIIA were identified by image segmentation involving thresholding and subsequent conversion of the images to a binary format. For calculating the width of cortical actin and MIIA, the sum of the pixels of high intensity around the perimeter of a cell was normalized to its contour length. The contour length was obtained via Canny edge detection of the binary images, followed by image dilation and thinning. For the integrated cortical signal intensity measurements, after using the same image thresholding and binary conversion to track the location of the high-intensity signals around the perimeter of the cells, the sum of those pixel intensities from the original images was acquired. These values were normalized to those of the first frame (t = 0). For the percentage increase of the width/integrated signal, the width/integrated signal of the first time point after stimulation was normalized to the values before stimulation (before medium addition).

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