LatB and CK-666 each impair Naegleria’s phagocytosis. (A) Cells were starved, treated with inhibitors or controls, and fixed as in Fig. 5. Then, the GFP intensities of 30,000 cells per condition were measured by flow cytometry. Cells were initially gated to select only intact, single cells. Then, an untreated control from each replicate (top row) was used to create a gate for GFP⁺ cells (dashed line). Histograms display the percentage of cells plotted against GFP intensities for ethanol- and LatB-treated cells (middle row) or CK-689– and CK-666–treated cells (bottom row). The histograms from replicate 2 are also shown in Fig. 5, with y axes set to the same scale. (B) Cells were starved and then mixed with GFP-expressing E. coli (shown in magenta) for 1 h. Cells were fixed and stained with Alexa Fluor-568–labeled phalloidin to detect F-actin (green) and imaged using widefield fluorescence microscopy. Maximum intensity projections (max. proj.; top) and single xy, zy, and xz slices are shown intersecting nonintact (white arrowheads) and intact (gray arrowheads) bacteria associated with representative cells. AU, arbitrary units.