Exosome-Mediated Regulation of EWS Cells

EWS cells (2 × 10⁴ cells/well) were seeded in a 6-well ultra-low adherent plate (Corning) and pretreated with exosomes (0 or 20 μg/mL) in a sphere assay. Following 5 days, cells were harvested and lysed for use in western blot. Total protein was isolated from cells and exosomes using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein from cells was quantified using the BCA method (Pierce, Waltham, MA, USA) and the amount of protein in exosomes was estimated by measuring protein content with the Bradford assay (Bio-Rad, Hercules, CA, USA). SDS-PAGE was conducted under reducing or non-reducing conditions followed by immunoblot as previously described [43 (link)]. Antibodies used in this experiment were TSG101, CD63, Calnexin (1:500, all Santa Cruz Biotechnology, Dallas, TX, USA), HIF-1α (1:1000, BD Biosciences, San Jose, CA, USA), CASP8AP2/FLASH (1:1000, Abcam, Cambrige, UK) and α-Tubulin (1:4000, Sigma Aldrich, St. Louis, MO, USA). Blots were incubated with appropriate horseradish peroxidase-tagged secondary antibodies and visualized using the _MYECL Imager (Thermo Fisher Scientific, Waltham, MA, USA).