Journal: Cell Discovery

Article Title: HCV-associated exosomes promote myeloid-derived suppressor cell expansion via inhibiting miR-124 to regulate T follicular cell differentiation and function

doi: 10.1038/s41421-018-0052-z

Figure Lengend Snippet: a Identification of CD63, CD81 and HSP90 expressions in exosomes isolated from the plasma of HCV patients and HS (with exoEasy Maxi Kit) by immunoblotting. b Evaluation of CD63 concentration in the plasma and purified exosomes isolated from the same subject, or in the supernatant and enriched exosomes with exoEasy Maxi Kit. The same amount of proteins were loaded in the immunoblotting. c Detection of CD81 expression in exosomes (purified by CD81-coated beads) by flow cytometric analysis. d Electron microscope observation of exosomes. Exosomes isolated from the supernatant of HCV +/− Huh7 cells by differential centrifugation were immuno-stained with or without anti-CD81 antibody, followed by secondary antibody labeled with 12 nm gold particles, then subjected to IEM observation. Arrowed structures represent exosomes without gold labeling (left panel) and exosomes associated with gold particles (right panel). Bars = 50 or 100 nm as indicated. e Detection of HCV RNA by RT-PCR in exosomes purified by sequential centrifugation from HCV +/− subjects (upper panel) and quantification of their copy numbers in exosomes isolated from 1 ml plasma of HCV-infected patients by real-time RT-PCR. f Detection of HCV RNA by real-time RT-PCR in exosomes isolated by the centrifugation method from culture supernatants of HCV +/− Huh7 cells. HCV RNA copy number, shown as IU/ml supernatant-derived exosomes, is shown in the upper panel. Repeated experiments by RT-PCR measuring HCV RNA in exosomes purified by CD81-coated beads from the culture supernatant of HCV-infected (Huh7R) and -uninfected (Huh7) hepatocytes are shown in the lower panel. g Detection of HCV Core protein in exosomes isolated from the plasma of HCV +/− subjects or from the supernatants of HCV +/− Huh7 cells by exoEasy Maxi Kit followed by CD81-coated beads purification methods. Lower panel show exosomes isolated from the supernatants of HCV +/− Huh7 cells by the sequential centrifugation method. Cell lysate from Huh7 and Huh7R hepatocytes served as negative and positive controls. h Repeated experiments by RT-PCR measuring HCV RNA in CD33 + myeloid cells incubated with exosomes isolated from the supernatants of HCV +/− Huh7 cells overnight

Article Snippet: After washing with PBS, the grids were stained with 1% aqueous uranyl acetate before observation with a Zeiss Auriga 40 operating in STEM mode at 30 kV electron microscope.

Techniques: Isolation, Clinical Proteomics, Western Blot, Concentration Assay, Purification, Expressing, Microscopy, Centrifugation, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Infection, Quantitative RT-PCR, Derivative Assay, Incubation