Septin depletion reduces acidification of GFP-CD63 endosomes, and inhibition of dynein motility enhances the percentage of LBPA-positive GFP-CD63. (A) Confocal microscopy sections of living MDCK cells after 36 h of transfection with pHluorin_M153R-CD63-mScarlet and scramble control or SEPT7 shRNA from EBFP2-expressing plasmid DNA. Scale bars, 10 μm. (B) Bar graph shows the mean (±SEM, error bars) percentage of mScarlet-labeled compartments that were free of GFP fluorescence per cell (n = 10–11). Data were analyzed with a Mann–Whitney U test (P < 0.01) and are representative of two independent experiments. (C) Spinning disk confocal microscopy images of living MDCK-GFP-CD63 cells imaged after 6 h of incubation with DQ Red BSA, which followed a 36-h transfection with plasmids co-expressing EBFP2 and control or SEPT7 shRNA. Scale bars, 10 μm (main panels, EBFP2 insets) and 5 μm (insets of outlined regions). (D) Bar graph shows the mean percentage (±SEM, error bars) of GFP-CD63 compartments that contained DQ Red BSA per cell (n = 20–22). Data were analyzed with a Mann–Whitney U test (P < 0.001) and are representative of two independent experiments. (E) Confocal microscopy sections of MDCK-GFP-CD63 cells, which were stained with an antibody against LBPA after 30 min of treatment with carrier (DMSO) or dynarrestin (10 μM). Insets show outlined regions of the perinuclear cytoplasm in higher magnification. Scale bars, 10 and 5 μm (insets). (F) Bar graph shows the mean percentage (± SEM, error bars) of GFP-CD63 endosomes that contain LBPA per cell (n = 11). Data were analyzed with an unpaired t test (P = 0.001) and are representative of two independent experiments. ** P < 0.01, *** P < 0.001.