Fig. 5: Autophagic cargos, ESCRT-III components, and Rab13 serve as distinct markers that differentiate AEVs from conventional exosomes.

A The vesicle fraction from supernatant of wild-type (WT) or gene knockout (KO) 293 T cells with or without starvation induction was collected and blotted for the indicated proteins. Similar results were obtained in three independent experiments. B–D The iodixanol density gradient procedure to separate sEV fraction secreted from serum-starved 293 T cells (C) and subcellular fractionations of these cells (D). A 5-40% Optiprep gradient was collected in 12 fractions (F1-F12) and analyzed by western blot. Similar results were obtained in three independent experiments. B Created in BioRender. Ruan, H. (2025) https://BioRender.com/al3dqi8. E Western blot of indicated proteins from untreated AEVs or AEVs incubated with 100 μg/ml trypsin and/or 1% Triton X-100 (TX-100) for 30 minutes at 4 °C. Similar results were obtained in three independent experiments. F Immunogold-stained electron microscopy images of AEVs from starved 293 T cells. The gold particles (black dots) indicated the presence of LC3B in the vesicles, while p62 and Rab13 were present on the vesicle membranes. Bar =100 nm. Similar results were obtained in three independent experiments. The AEV fractions from THP-1 (G) and Neuro−2a (H) cell lines under normally cultured or serum-starved condition were collected and blotted for the indicated proteins. Cell lines were cultured in serum-free medium to induce autophagy. Similar results were obtained in three independent experiments. I Schematic of distinct biomarkers for classic exosomes and AEVs, both of which were two subtypes of small EVs. Source data are provided as a Source Data file.