Figure 4.

Autophagy cargo receptors are secreted as EVP-associated proteins in response to lysosome inhibition. (A) Cell lysate (WCL) and fractionated conditioned media (CM) were collected from serum-starved HEK293Ts treated with vehicle or 20 nM BafA1 for 16 h. CM subjected to serial differential ultracentrifugation to recover large EVs (10,000 g; 10K), small EVPs (100,000 g; 100K), and precipitated free soluble protein (TCA). Equal protein from WCL and fractionated CM were probed for the indicated targets (n = 3). (B) Quantification of LC3 and autophagy cargo receptors in the indicated fractions of CM from serum-starved BafA1 treated cells relative to WCL (mean ± SEM; n = 3; ***, P < 0.005). Statistical significance between CM fractions was calculated by nonparametric one-way ANOVA with Dunnett’s post hoc test. (C) Small EVPs from CM separated via linear sucrose density gradient ultracentrifugation, fractionated, and blotted to detect LC3, autophagy cargo receptors, and CD9 (n = 3). (D) Percent of total secreted LC3, cargo receptors, and CD9 detected in gradient fractions (mean ± SEM; n = 4). (E) Representative blots of indicated proteins from untreated EVPs or EVPs incubated with 100 μg/ml trypsin and/or 1% Triton X-100 (TX-100) for 30 min at 4°C (n = 3). (F) Percent protease protection for indicated proteins in EVPs incubated with 100 μg/ml trypsin and/or 1% TX-100 (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01; ****, P < 0.001). Statistics calculated by nonparametric one-way ANOVA with Dunnett’s post hoc test. (G) Representative TEM of small EVPs collected from vehicle or 20 nM BafA1 treated cells immunostained with anti-p62 primary antibody and detected using ultrasmall gold-conjugated secondary antibody with silver enhancement (scale bar, 500 nm). Source data are available for this figure: SourceData F4.