CK-666 treatment enhances the relative concentration of cortical to intracellular F-actin. (A) 15 amoebae treated with the Arp2/3 complex inhibitor CK-666 from the experiments described in Fig. 3 were imaged using widefield fluorescence microscopy. A line with a width of 50 px, representing 3.25 µm, was drawn perpendicular to the cell edge on a random, relatively straight part of each cell in a Z plane in which the cell edge was in focus. Panels i–iii: examples of these regions on cells are shown, with one example from each of the three biological replicates analyzed. Panel iv: the regions of interest of six additional examples are shown. (B) The pixel intensities along the long axis of each box shown in A were normalized to the average pixel intensity of an area inside the cell, which was set to 1. These normalized intensities were plotted against the distance, with 0 indicating the cell edge (dashed line). The horizontal line indicates the maximum fluorescence intensity at the cell edge. The first three panels (i–iii) correspond to the images in A i–iii, and the rightmost “all” panel depicts all the data used to generate the plot in Fig. 3 J. (C and D) Images mirror A and B, respectively, using the inactive control CK-689. AU, arbitrary units.