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

Figure 1. p97 is a direct and essential component of the lysosomal damage response.

Figure 1

  1. HeLa cells expressing LAMP1‐RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin‐3 (Gal3) antibodies, and analyzed by confocal microscopy.
  2. p97 translocates to ruptured lysosomes. Cells co‐expressing LAMP1‐RFP and GFP‐p97 were treated as in (A) and stained with the pan‐ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD). Student's unpaired t‐test.
  3. Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD). Student's unpaired t‐test.
  4. Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout, and stained for endogenous Gal3. Quantification of the number of Gal3‐positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥ 30 cells quantified per condition (one‐sided Welch's unequal variance t‐tests comparing the siCtrl with sip97).
  5. Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM), or the p97 inhibitor NMS‐873 (5 μM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition (one‐sided Welch's t‐tests).
  6. p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97, or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.
  7. Tau fibrils are endocytosed and induce lysosomal damage. Tau fibrils were generated in vitro, fluorescently labeled by Dylight 488, and fragmented by sonication (488‐labeled tau). HeLa cells expressing mCherry‐Gal3 were incubated with 300 nM tau fibrils for 24 h. Arrows indicate compartments that contain 488‐labeled tau and are positive for mCherry‐Gal3 indicating lysosome rupture. Percentage of cells with Gal3‐positive vesicles was quantified from three independent experiments (mean ± SD). Student's unpaired t‐test.
  8. Accumulation of tau‐induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3‐positive vesicles was quantified from three independent experiments (mean ± SD). Student's unpaired t‐test.
Data information: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 10 μm.