Skip to main content
. 2019 Aug 21;20(10):e48014. doi: 10.15252/embr.201948014

Figure EV1. UBE2QL1 translocates specifically to damaged lysosomes (related to Fig 1).

Figure EV1

  1. Immunofluorescence analysis of UBE2QL1‐HA in cells with depolarized mitochondria. HeLa cells transiently transfected with mCherry‐Parkin and UBE2QL1‐HA were treated with 10 μM Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) or DMSO alone (untreated) for 4 h, fixed and stained with antibodies specific for the mitochondrial protein Tom20 and HA. Note that there is no translocation of UBE2QL1‐HA to depolarized mitochondria. Scale bar: 10 μm.
  2. HeLa cells that were transfected with control (Ctrl) or UBE2QL1‐targeting siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 3 h were processed for immunofluorescence microscopy with antibodies against endogenous UBE2QL1 and LAMP1. Note that the UBE2QL1 signal colocalizing with LAMP1 in LLOMe‐treated cells is suppressed by UBE2QL1 depletion indicating its specificity. Arrows indicate colocalizing vesicles. Scale bar: 10 μm.
  3. Automated quantification of (B). Percentage of cells with more than 3 UBE2QL1‐positive LAMP1 vesicles. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). ***P < 0.001; ****P < 0.0001 (one‐way ANOVA with Bonferroni's multiple comparison test).
  4. UBE2QL1 localizes to late endosomes/lysosomes damaged by endocytosed tau fibrillar tangles. HeLa cells stably expressing mCherry‐Gal3 were incubated with Alexa488‐labeled tau fibrils for 1 day. Cells were fixed and stained with indicated antibodies to UBE2QL1 and LAMP1 to detect endogenous UBE2QL1 on damaged lysosomes. Note colocalization of UBE2QL1 with tau‐containing LAMP1 vesicles that are positive for Gal3 indicating damage. Arrow indicates colocalizing vesicle. Scale bar: 10 μm.