Prolonged lysosome damage induces the extracellular release of LAMP1-positive structures. (A) MEF cell line was treated with or without 2 mM LLOMe for 20 h and then stained with LAMP1 and WGA. The marked regions were enlarged and demonstrated on the right. Scale bar denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (B) Statistic result of the rate of MEF cells with extracellular LAMP1 signal when treated with different concentrations of LLOMe for 22 h (related to Fig. S1 A). The result represented for three independent replicates. 40–50 cells were counted each time and 140–150 cells were analyzed in total for each group. (C) MEF cell line was treated with 2.5 mM LLOMe for 22 h and then fixed and stained by FM 4-64. Scale bar denoted 20 μm for the large-view graph and 5 μm for the small-view graph. (D) TSPAN4-BFP MEF stable cell line was treated with SiO₂ (1 mg/ml), GPN (150 μM), and MSU (10 mg/ml), respectively, for 22 h. Then stained with LAMP1. The marked regions were enlarged and demonstrated on the right. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. (E) Time-lapse imaging of TSPAN4-mCherry LAMP1-GFP stably expressing MEF cells treated with 2 mM LLOMe. Cells had been treated with LLOMe for 8 h before the first frame was captured. The last frame was captured >5 h later. The marked regions were enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graphs, 5 μm for the small-view graphs. The complete process could be observed in Video 1. (F) CLEM data of TSPAN4-mCherry LAMP1-GFP stably expressing MEF cells treated with LLOMe for 22 h. The marked region in the fluorescent graph was enlarged and demonstrated on the right, stacked with the electron microscope graph. Four marked regions were enlarged and demonstrated with electron microscope graphs. Scale bars denoted 20 μm for the large-view fluorescent graph, 5 μm for the small-view fluorescent graph and 500 nm for all the electron microscope graphs.