Figure 3.
Extracellular release of LAMP1-positive structures required the fusion of autolysosomes and the basal plasma membranes. (A) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h and then stained with LC3. The marked region in the large graph was enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (B) Time-lapse imaging of GFP-LC3 LAMP1-mCherry stably expressing MEF cells treated with 2 mM LLOMe and stained with Cy5. The first frame was captured about four and a half hours after LLOMe addition. The marked regions were enlarged and demonstrated on their right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process can be observed in Video 6. (C) Control, FIP200−/−, Atg3−/− and Atg5−/− MEF cells were treated with 2.5 mM LLOMe for 22 h then fixed and stained with FM 4-64. The representative images of each group were converted into gray-value mode. The scale bar denoted 20 μm. (D) Western blot results of the samples from experiments in C. Knocking Out efficiency of FIP200, Atg3 and Atg5 was verified respectively. (E) Statistic results of the area of extracellular lysosomal structure of FIP200−/−, Atg3−/− and Atg5−/− MEF cells in C. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001. (F) Control, Syntaxin17−/− and mCherry-Syntaxin17 Rescue 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. (G) Western blot result of the samples from experiments in F. Knocking Out and rescue efficiency of Syntaxin17 was verified. (H) Statistic results of the area of extracellular lysosomal structure of groups in F. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001, NS = not statistically significant. (I) Control, Sec22b−/− and GFP-Sec22b Rescue; Control, Syntaxin4−/− and GFP-Syntaxin4 Rescue; Control, SNAP23−/− and GFP-SNAP23 Rescue 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. (J) Western blot results of the samples from experiments in I. Knocking Out and rescue efficiency of Sec22b, Syntaxin4 and SNAP23 was verified respectively. (K) Statistic results of the area of extracellular lysosomal structure of groups in I. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001, NS = not statistically significant. Source data are available for this figure: SourceData F3.

Extracellular release of LAMP1-positive structures required the fusion of autolysosomes and the basal plasma membranes. (A) TSPAN4-BFP stably expressing MEF cell line was treated with 3 mM LLOMe for 22 h and then stained with LC3. The marked region in the large graph was enlarged and demonstrated beneath. Scale bars denoted 20 μm for the large-view graph, 5 μm for the small-view graphs. (B) Time-lapse imaging of GFP-LC3 LAMP1-mCherry stably expressing MEF cells treated with 2 mM LLOMe and stained with Cy5. The first frame was captured about four and a half hours after LLOMe addition. The marked regions were enlarged and demonstrated on their right. Scale bars denoted 20 μm for the large-view graphs, 2 μm for the small-view graphs. The complete process can be observed in Video 6. (C) Control, FIP200−/−, Atg3−/− and Atg5−/− MEF cells were treated with 2.5 mM LLOMe for 22 h then fixed and stained with FM 4-64. The representative images of each group were converted into gray-value mode. The scale bar denoted 20 μm. (D) Western blot results of the samples from experiments in C. Knocking Out efficiency of FIP200, Atg3 and Atg5 was verified respectively. (E) Statistic results of the area of extracellular lysosomal structure of FIP200−/−, Atg3−/− and Atg5−/− MEF cells in C. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001. (F) Control, Syntaxin17−/− and mCherry-Syntaxin17 Rescue 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. (G) Western blot result of the samples from experiments in F. Knocking Out and rescue efficiency of Syntaxin17 was verified. (H) Statistic results of the area of extracellular lysosomal structure of groups in F. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001, NS = not statistically significant. (I) Control, Sec22b−/− and GFP-Sec22b Rescue; Control, Syntaxin4−/− and GFP-Syntaxin4 Rescue; Control, SNAP23−/− and GFP-SNAP23 Rescue 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. (J) Western blot results of the samples from experiments in I. Knocking Out and rescue efficiency of Sec22b, Syntaxin4 and SNAP23 was verified respectively. (K) Statistic results of the area of extracellular lysosomal structure of groups in I. Error bars denoted ± SEM from three independent fixed-cell imaging experiments, 50 cells were analyzed per experiment and 150 cells were analyzed in total for each group. T test, ****P < 0.0001, NS = not statistically significant. Source data are available for this figure: SourceData F3.

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