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. 2018 Oct 12;37(21):e99753. doi: 10.15252/embj.201899753

Figure 4. ESCRTs are essential for the repair after endolysosomal damage.

Figure 4

  1. HeLa cells stably expressing CHMP4B‐eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND‐99 and fixed at different time points as indicated. After 10 min of LLOMe treatment, CHMP4B is recruited whereas the number of Lysotracker‐positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm.
  2. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker‐positive foci per cell at different time points. Error bars correspond to 95% confidence intervals.
  3. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101‐ and ALIX‐depleted cells. HeLa cells stably expressing CHMP4B‐eGFP were co‐transfected with siRNAs against TSG101 and ALIX. Forty‐eight hours post‐transfection, cells were pre‐treated for 20 min with 75 nM Lysotracker Deep Red, which was used as a read‐out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live‐cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.