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. 2016 Oct 17;36(2):135–150. doi: 10.15252/embj.201695148

Figure EV1. p97 is a direct component of the lysosome damage response (related to Fig 1).

Figure EV1

  1. LLOMe ruptures lysosomes and induces an autophagic response. HeLa cells were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with LAMP1 and LC3 antibodies, and analyzed by confocal immunofluorescence microscopy.
  2. Endogenous p97 translocates to ruptured lysosomes. Cells transiently expressing LAMP1‐RFP were treated as in (A) and stained for endogenous p97 and p62.
  3. Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition from Fig 1D.
  4. Cells were treated with 10 nM bortezomib (Btz) or with vehicle alone (DMSO) for 12 h and ubiquitin conjugates (Ub) in whole‐cell lysates were detected by immunoblotting.
  5. Tau fibrils are endocytosed and induce autophagy. Tau fibrils were generated in vitro, fluorescently labeled by Dylight 488, and fragmented by sonication (488‐labeled tau). HeLa cells were incubated with 300 nM tau fibrils for 24 h, fixed, and stained with LC3 and LAMP1 antibodies. Untreated cells served as a control. Quantification of the fraction of LC3‐positive LAMP1 vesicles per cell.
Data information: Data represent mean ± SD from three independent experiments. **P < 0.01 (one‐sided Welch's t‐tests). Scale bars, 10 μm. Source data are available online for this figure.