Neither cell morphology nor actin polymer content is affected by jasplakinolide (Jasp), phalloidin (Phall), or cytochalasin (CytoD). (A) Cells were incubated in media ± inhibitors or controls for 10 min, then fixed and stained with Alexa Fluor-488–labeled phalloidin to detect F-actin (green) and DAPI to label DNA (magenta) before imaging using widefield fluorescence microscopy. Representative cells are shown. (B–G) Amoebae treated as in A were stained only with Alexa Fluor-488–labeled phalloidin (with the exception of an unstained control, shown in B) before analysis by flow cytometry. Representative histograms of F-actin staining intensity compare drug treatments with respective controls for one of three biological replicates. (H) Average intensities calculated from E–G were normalized to the stained control (B), which was set to 1 (dashed line). Each point represents the average normalized fluorescence intensity of F-actin staining (experimental replicates are coordinated by shape), and lines indicate the mean of three experimental replicates ± SD. Statistical significance was determined using an ordinary ANOVA followed by Tukey’s multiple comparison test. (I) Images like those shown in A were analyzed with the dataset presented in Fig. 3. Values are averages (Avg) ± SD from three independent replicates, each of which encompassed 50 cells per treatment condition. No statistical differences were found comparing jasplakinolide, phalloidin, or cytochalasin D treatments with controls using an ordinary ANOVA followed by Tukey’s multiple comparison test. *The percentage of cells with actin spheres was calculated for one representative dataset and, due to large variability between control samples, was not quantified for the remaining datasets. AU, arbitrary units; RFU, relative fluorescence units; SM+CK, SMIFH+CK-666 combined treatment.