Short treatments of AntA do not stop the movement of Rab5A-labeled endosomes or lysosomes. (A) Kymograph of Rab5 motility in cultured hippocampal neurons expressing mEmerald-Rab5a and mCherry2. Cells were treated for 1 h with either 4 nM AntA or vehicle and then imaged. Imaging was performed in phenol red–free Hibernate E media to reduce background fluorescence. (B) Quantification of the Rab5 movement from axons as in Fig. 1 A. n = 8–10 axons per treatment from four independent animals. (C) Quantification of lysosome movement in fibroblasts. Cells were imaged live in galactose media after 1 h of treatment with vehicle or 40 nM AntA. LysoTracker Red DND-99 was used to visualize lysosomes. TrackMate 7 was used to track and quantify the total distance traveled by each lysosome track. (D) Quantification of the lysosomal confinement ratio from images acquired as in C. The confinement ratio of each lysosome track is defined as the net distance traveled divided by the total distance traveled. The net distance is defined as the distance between the starting and ending positions of the particle. The total distance traveled is the sum of the tracked segments. For all graphs, scale bars = 20 μm, and bars on boxplots show the 10–90th percentile. P values for B–D were calculated by two-tailed, unpaired t tests with Welch’s correction.