Related to Fig. 3 . (A) MEF shows the process of image analysis during statistics of extracellular lysosome structures marked with FM 4-64. MEF cells were treated with 2.5 mM LLOMe for 22 h then fixed with 4% PFA and stained with FM 4-64. Images were captured at two layers (Z = 0.0 μm and Z = 1.0 μm, A1 and A2), then stacked (A3) and converted into gray-value mode (A4). The extracellular region were marked in ImageJ FIJI (A5) and then the threshold was set to the baseline of signal intensity on retraction fibers and plasma membranes (A6). (B) Control, SNAP29^−/−, and Syntaxin3^−/− MEF cells were treated with 2.5 mM LLOMe for 22 h then fixed and stained with FM 4-64. The representative images were converted into gray-value mode. The scale bar denoted 20 μm. (C) Statistic results of the area of extracellular lysosomal structure of groups in B. Error bars denoted ± SEM from only one fixed-cell imaging experiment, 50 cells were analyzed for all groups. T test, NS = not statistically significant. (D) Western blot results of the samples from experiments in B. Knocking out efficiency of SNAP29 and Syntaxin3 was verified respectively. Source data are available for this figure: SourceData FS3.