Vcl KO increases GTP-RhoA–mediated ROCK activity and cell blebbing in MDA-MB-231 cells. (A) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces, where each outline represents a 4-s interval. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. (B) Representative time lapse indicating that blebs are forming in the Vcl KO cells as shown by separation of the membrane from the actin cortex labeled using MemGlow and LifeAct, respectively. Scale bar, 10 µm in the first image. Scale bar, 5 µm in the zoomed-in time-lapse images. (C) Representative phase-contrast images of cells treated with 20 µm Y-27632 (Y27) or the vehicle control (deionized water). Scale bar, 100 µm. (D) Representative color-coded time lapse of MDA and Vcl KO cell shape on 2D surfaces treated with 20 µm Y-27632 or the vehicle control. Scale bar, 20 µm in the image with the entire cell. Scale bar, 5 µm in the inset images in red boxes. For D, each outline represents a 4-s interval. (E) Quantification of the percentage of blebbing cells (N = 3, n = 28–31 fields of view) and the number of blebs per minute per cell (N = 3, n = 36 cells) for cells treated with 20 µm Y-27632 or the vehicle control. (F) Quantification of the percentage of blebbing cells in cells treated with 25 µm Blebb or the vehicle control (N = 3, n = 29 fields of view). (G) Normalized GTP-loaded RhoA measured using absorbance at 490 nm for cells treated with 20 µm Y-27632 or the vehicle control (N = 4). Bar graphs denote the mean ± SEM. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.