Fig. 3

Inhibition of autophagosome/lysosome fusion induces retraction fiber and migrasome (R&M) formation. A Live-cell imaging for observing R&Ms formed in LN229 cells. Cells were treated with chloroquine (CQ; 50 μM, 12 h) or bafilomycin A1 (BafA1; 50 nM, 12 h). Gray, EGFP-CD9. Scale bars, 20 μm. B Quantification of the number of retraction fibers (RFs) per a cell. Image analyses were performed using results from (A). n = 19 for vehicle-treated condition, n = 18 for CQ- or BafA1-treated condition. C Quantification of the number of migrasomes per RF (100 μm). Image analyses were performed using the results from (A). D Tandem-fluorescent LC3B was expressed in LN229 cell. Fluorescent signal observed in both cell body and migrasome was quantified respectively. Cells were treated with CQ. Red/green, mRFP-EGFP-LC3B. Black, DiD lipophilic tracer. Scale bars (black), 20 μm. Scale bars (white), 10 μm. E Quantification of D. Total RFP⁺ vesicle area was quantified in each condition. F Hypothetic graphical scheme of the relationship between autophagosome/lysosome fusion and R&M formation. G In situ proximity ligation assay (PLA) was performed to examine the interaction between LC3B and ATG5 in migrasomes of LN229. Cells were treated with CQ. Red, PLA signal. Gray, EGFP-CD9. Scale bars, 20 μm. Scale bars in cropped panels, 10 μm. H Live-cell imaging for observing R&M formed in LN229 cells expressing EGFP-CD9 after transfection of STX17 or SNAP29 siRNAs. Gray, EGFP-CD9. Scale bars, 20 μm. I Quantification of the number of migrasomes per RF (100 μm). Image analyses were performed using the results from (H). n = 10 for each image. The unpaired nonparametric Mann–Whitney U-test was used to analyze the statistical significance between each group. Data are expressed as mean ± SEM. In all data, **indicates p < 0.01, and ***indicates p < 0.001. The figure is representative of three-biological replicates with similar results