(A) HeLa cells were incubated with BSA coupled to 5 nm gold for 10 min before being washed several times with PBS to remove any uninternalised label. The cells were then chased in full medium for either 30 min or 4 hr to load BSA-gold into endosomes or lysosomes respectively, then treated with BafA1 (100 nM, 16 hr), fixed, and prepared for conventional EM. Gold could be seen associated with extracellular vesicles from the cells chased for 30 min, but not from the cells chased for 4 hr. Insets: monomeric gold could be found within MVBs from cells chased for 30 min (arrows), and aggregated within lysosomes from cells chased for 4 hr (arrowhead). Scale bar: 200 nm. (B) DMSO- or BafA1-treated HeLa cells (100 nM, 16 hr) were surface-labelled with an antibody against the CD63 lumenal domain to identify exosomes. Scale bar: 500 nm. (C) HeLa cells were treated with DMSO or BafA1 (100 nM, 16 hr) before being fixed and prepared for conventional EM. Exosomes were often associated with clathrin-coated pits (arrow). Scale bar: 500 nm (D) The number of exosomes per μm of plasma membrane was quantified. Data shown are means from three independent experiments, ± S.E.M. ***p<0.001 (E) Culture supernatants were collected from mock-treated and BafA1-treated HeLa cells (100 nM, 16 hr), and centrifuged first at 10,000xg and then at 100,000xg. Western blots of the pellets were probed with anti-CD63. (F) Total internal reflection fluorescence microscopy (TIRF) was performed on HeLa cells transiently expressing both CLC-mCherry and CD63-GFP, following BafA1 treatment (100 nM, 16 hr). Representative stills are shown. Scale bar: 10 μm. (G) Conventional EM of BafA1-treated cells (100 nM, 16 hr), with arrowheads indicating proteinacious material on exosomes. Scale bars: 500 nm. See also Figure 2—figure supplement 1, 2.