Fig 1.
A) The construct of the plasmid in Fig 1B is illustrated. B) 293T cells were transfected with the indicated plasmids, and the HiBiT signal in EVs and the whole cell lysate were compared. The data are the averages (±SD) of three experiments. C) Purified EVs from 293T cells transfected with HiBiT-EGFP-CD9 were added to the indicated cells. After 12 h of incubation, total cellular association and cytosolic delivery were measured. The endosomal escape efficiency was calculated as follows: Cytosolic delivery/the average of total cellular association. In this experiment, the comparison of EVs purified via UF or UF-SEC was performed. The averages (±SD) of three experiments are displayed.
Fig 2.
A) The structures of CD9 and short CD9 are illustrated. SEL; short extracellular loop, LEL; long extracellular loop, ICL; intracellular loop. B) 293T cells in 6-well plates were transfected with EGFP-CD9 or EGFP-sCD9. After 24 h of transfection, the culture medium was exchanged, and the cells were further incubated for the indicated time. EVs from the culture supernatant were purified and diluted to 2 ml (initial volume), and the HiBiT signals were measured. C) Purified EVs collected in B) were added to 293T-LHA cells. After 12 h of incubation, cytosolic delivery and total cellular association were measured.
Fig 3.
A) The cell-penetrating peptides and fusogenic peptides inserted into sCD9 are shown. B) 293T cells in 6-well plates were transfected with the indicated plasmids in A), followed by medium exchange after 24 h. EVs were collected after another 48 h of incubation and diluted to 2 ml, and then the luminescence was measured to quantify the HiBiT-tagged protein level. The data are the averages (±SD) of three experiments. C–E) The EVs purified in B) were added to 293T-LHA cells. After 12 h of incubation, cytosolic delivery and total cellular association were measured. The endosomal escape efficiency was calculated as in Fig 1C. The data are the averages (±SD) of three experiments.
Fig 4.
A–B) 293T cells stably expressing acylation-tagged EGFP were transiently transfected with the indicated plasmids (see S4B Fig in S1 File), and the extracellular vesicles were purified from culture medium as in Fig 3B and were added to 293T-LHA or HeLa-LHA cells. Cytosolic delivery and total cellular association were measured after 12 h of incubation.
Fig 5.
A–B) 293T cells expressing HiBiT-EGFP-sCD9 under a doxycycline-inducible promoter and 293T-LHA or HeLa-LHA cells were cocultured for 48 h via Transwell (S5A Fig in S1 File), and the cytosolic delivery in the receptor cells was measured by luminescence. C–D) Cells in A and B were cocultured in the same well for 24 h, and the cytosolic delivery was measured by luminescence. E–F) The experiment in C) was performed under the treatment of 10nM or 20nM of EIPA. For toxicity, EIPA was added 12 h after seeding (12-h treatment).