Phagocytosis is less efficient following Arp2/3 complex inhibition. (A) Amoebae were starved for 1 h, then incubated with controls or inhibitors and GFP-expressing E. coli for 45 min. Cells were then fixed and imaged. GFP intensity is shown using an inverted LUT (lookup table; top) and in magenta (bottom). (B) Cells were treated as in A, then the GFP intensities of 30,000 cells/condition were measured by flow cytometry. Representative histograms display the percentage of cells plotted against GFP intensities for ethanol- and LatB-treated cells (left) or CK-689– and CK-666–treated cells (right). Plots are from one of three experimental replicates (also see Fig. S9). (C) The median fluorescence intensity was calculated from experiments shown in B. For each replicate (coordinated by shape; circles, triangles, and squares), fluorescence intensity of control and treated cells was normalized to a buffer-only control. (D) The population of buffer-only control cells was used to gate a GFP-positive population for each experimental replicate. Each point represents the percentage of cells falling within the GFP-positive gate. For graphs in C and D, lines indicate the mean of three experimental replicates (which are coordinated by shape) ± SD. The dashed lines are set to the average from the control sample. Statistical significance was determined using an ordinary ANOVA and Tukey’s multiple comparison test. AU, arbitrary units; RFU, relative fluorescence units.