Fig 1: Extracellular vesicles released by HSC-3 cells have characteristics of exosomes and carry pain and metastasis genes as cargo. (a) Representative transmission electron micrographs of EVs isolated from conditioned media of HSC-3. Images were taken at 45,000× (left) and 92,00× (right) magnification. Isolated vesicles display cup-shaped morphology and membrane layer characteristic of exosomes (arrows). (b) The diameters of vesicles from HSC-3 were measured on micrographs taken at 92,000× (4–6 fields). (c) Representative nanoparticle tracking analysis (NTA) of the distribution of measured EV hydrodynamic diameter (mode indicated above the trace). Extracellular vesicle sizes as measured from electron micrographs and by NTA are typical of exosomes. (d) Isolated EVs express exosome endocytic marker proteins TSG101 and ALIX, and tetraspanin, CD63. Calnexin (CANX, endoplasmic reticulum marker) and GM130 (cis-Golgi network marker) were not detected. Exosome samples were obtained from cells grown under normoxic conditions. Pain and metastasis gene proteins, MMP1 and THBS1 were detected in the HSC-3 EVs.