Skip to main content
. 2019 Jul 3;7:118. doi: 10.3389/fcell.2019.00118

FIGURE 1.

FIGURE 1

EELSs in COG KO cells are highly acidic and the activity of vacuolar ATPase is necessary for their long-term stability. (A) Specific accumulation of EELSs in COG KO cells. DIC images of HEK293T WT, COG4 KO, and stably rescued COG4 KO cells. (B) EELSs are highly acidic. HEK293T COG KO cells were incubated with LysoSensor Yellow Blue DND-160 as described in “Materials and Methods,” treated with vacuolar ATPase inhibitor Baf A1 and imaged with Zeiss LSM880. Note that before Baf A1 treatment, LysoSensor fluorescence is seen in the EELSs (arrows) due to a low pH environment within their lumen. After treatment with Baf A1 LysoSensor fluorescence is diminished. Scale bars are 10 μm. (C) Phase–contrast images of COG4 KO cells before and after drug treatment. EELSs disappear after 24 h of treatment with 200 nm Bafilomycin A1. Scale bars are 50 μm. (D) Bar graph indicates the average number of EELSs per cell before and after treatment with Baf A1. (E) COG depleted cells are more sensitive to Baf A1 treatment. WT and COG4 KO cells were seeded at 40% confluence and incubated in culture media with or without with Baf A1. 72 h later dishes were rinsed with PBS to remove dead detached cells and remaining cells were counted. Arrows point to EELSs that are between 1 to 10 μm in diameter. Three fields were imaged and error bars indicate SD for n = 3. ∗∗p < 0.01.