a, Whole cell (WCL) and EV lysates from cells treated in the absence or presence of 5μM GW4869 for 24h and immunoblotted for the indicated marker proteins (n=2 biologically independent experiments). b, Nanoparticle counting for a representative experiment in Panel a (line=mean; n=1, 3 technical replicates). c, Whole cell (WCL) and EV lysates harvested from equal numbers of HEK293T cells stably expressing non-targeting (NT), ATG7 or nSMase2 (nSM2) shRNAs were immunoblotted for indicated proteins (n=3 biologically independent experiments). d, Quantification of indicated protein levels in EVs from equal numbers of stable knockdown cells in Panel c relative to non-targeting (NT) shRNA (mean ±s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by one-way analysis of variance (ANOVA) coupled with Tukey’s post hoc. e, HEK293T cells co-transfected with FLAG-tagged FAN and myc-tagged LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2) or myc-BirA* were lysed, immunoprecipitated (IP) with anti-myc antibody, and immunoblotted (WB) with indicated antibodies (n=2 biologically independent experiments). f, Whole cell (WCL) and extracellular vesicle (EV) lysates harvested from equal numbers of HEK293T cells stably expressing non-targeting (NT), ATG7 or FAN shRNAs were immunoblotted for indicated proteins (n=3 biologically independent experiments). g, Quantification of indicated protein levels in EVs from equal numbers of stable knockdown cells in Panel c relative to non-targeting (NT) shRNA (mean ± s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. h, Domain map and primary LC3-interaction region (LIR) in FAN. i,, Cells co-transfected with FLAG-tagged FAN and myc-tagged LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2) or myc-BirA* were lysed, immunoprecipitated (IP) with anti-myc antibody, and immunoblotted (WB) with indicated antibodies (n=2 biologically independent experiments). j, Whole cell lysate (WCL) and EV fractions from cells stably co-expressing non-targeting (NT) or FAN shRNA along with FLAG-tagged wild-type FAN (WT) or mutant FAN (F602A) were immunoblotted for indicated markers (n=2 biologically independent experiments). k, Quantification of indicated proteins in EVs from equal numbers of FAN knockdown HEK293T cells expressing FLAG-tagged wild-type FAN (WT) versus mutant FAN (F602A) (mean ± s.e.m.; n=3 biologically independent experiments). Statistical significance calculated by paired two-tailed t-test. Data and unprocessed blots available in Source Data Fig. 7.