Fig 1: Effect of Vps4, Tsg101, or Alix proteins on particle budding. (A) HEK293T (4 × 106) cells were transfected with siVPS4 or siControl 6 h before being transfected with 3HA-BEnv or 3Flag-BGag (BFV Gag protein), and then cultured for 24 h. The cell culture supernatants were filtered through a 0.45 µm filter and purified by ultracentrifugation. Transfected cells were lysed using lysis buffer. Levels of proteins in cells and supernatants were measured using Western blot. (B,F) To quantify the levels of released VLPs, the amount of BGag in VLPs was normalized against the amount of intracellular BGag, which was first normalized against the GAPDH loading control. Mean values and the standard deviation of particle-associated BGag protein corrected for intracellular expression levels (n = 3) are shown. The data are the averages of three independent experiments. Compared with the siControl: ** p < 0.01. (C) HEK293T (4 × 106) cells were transfected with 3HA-BEnv, 3FlagBGag, and either the 3HA, 3HA-Alix, or 3HA-Tsg101 vector constructs and harvested at day 2 post-transfection. The cell culture supernatants were filtered through a 0.45 µm filter and purified by ultracentrifugation. Transfected cells were disrupted using lysis buffer. Levels of proteins in cells and supernatants were measured using Western blotting. (D) HEK293T (4 × 106) cells were transfected with siAlix or siControl 6 h before transfected with 3HA-BEnv, 3Flag-BGag, and either the pCMV-3HA or pCMV-3HA-Alix vector constructs, and then cultured for 24 h. The cell culture supernatants were filtered through a 0.45 µm filter and purified by ultracentrifugation. Levels of proteins in cells and supernatants were measured using Western blotting. (E) HEK293T (4 × 106) cells were transfected with siTsg101 or siControl 6 h before transfected with 3HA-BEnv, 3Flag-BGag, and either the pCMV-3HA or pCMV-3HA-Tsg101 vector constructs, and then cultured for 24 h. The cell culture supernatants were filtered through a 0.45 µm filter and purified by ultracentrifugation. The levels of released VLPs were quantified according to the method described in the statistical analysis section.
Fig 2: Nuclear exosomes HMGB3 derived from the micronucleus can be transferred from CNE2 to HUVECs and is associated with NPC metastasis.A Immunofluorescence (IF) images of NP69 with or without HMGB3 overexpression. The right images show a magnified nucleus and MN. The scale bar represents 50 µm. B IF images of NP69 oeHMGB3 were stained by HMGB3 (green), CD63 (red), and nucleus (blue). The scale bar represents 20 µm. C CNE2 cells transfected with fluorescent GFP-labelled HMGB3 overexpresses lentivirus were cocultured with HUVECs in a transwell Chamber. The scale bar represents 50 µm. D Western blotting analysis of the expression of HMGB3 in exosomes from NPC serum and CNE2-CM (cell medium). E Transmission electron microscopy of exosomes derived from NPC serum and CNE2-CM. The scale bar represents 200 nm. The red boxes indicate regions of pictures shown in the bottom two pictures. F Nanoparticle tracking analysis showed the size and distribution of exosomes isolated from NPC serum and CNE2-CM. G HUVECs uptake of exosomes released by NPC with a confocal microscope. blue: Hoechst staining; red: PKH26-labelled exosomes; light blue: DRAQ5 dye-labelled DNA. H Western blotting analysis of Alix, CD9, actinin-4 and flotillin-1 in NPC exosomes and CNE2 cells. I Detection of HMGB3 in serum exosomes of NPC patients and normal persons by Western blot. J–L Flow cytometry analysis showed the percentage of gDNA and HMGB3 expression in exosomes of NPC patients and normal persons. J: normal persons; K: NPC patients without metastasis; L: NPC patients with metastasis.
Fig 3: Characterisation of separated EVs by immunoelectron microscopy. Immuno-localisation of tetraspanins (CD9, CD63) on mEVs (A) and sEVs (D). EV markers Alix (E) and TSG101 (F) are present on sEVs. Identification of NME1 and NME2 on mEVs (B,C). NME1 cannot be detected on sEVs (G), whereas NME2 staining can be observed on sEVs (H). Gold particles are indicated by white arrowheads (10 nm) and black arrow (20 nm). mEVs and sEVs stained positive for EV markers CD63, CD9, Alix and TSG101 (A,D–F) were isolated from MDA-MB-231T cell line expressing the control vector pCDNA. NME1-positive mEVs (B) and NME1-negative sEVs (G) were isolated from F::NME1 overexpressing cells, whereas NME2 positive mEVs (C) and sEVs (H) are derived from M::NME2 overexpressing MDA-MB-231T cells.
Fig 4: Detection of NME1 and NME2 in extracellular vesicles (EVs) derived from supernatants of NME1 or NME2 overexpressing, and control vector expressing MDA-MB-231T cell lines with WESTM Simple. Abbreviations: F::NME1, M::NME2 and Co stand for protein or EV lysates from FLAG::NME1, MYC::NME2 and control vector-transfected cells, respectively. Small- and medium-size extracellular vesicles are abbreviated as sEV and mEV, respectively. (A) Vesicle marker detection in EV lysates. TSG101 content of sEV fraction was higher than that of the mEV fraction. The enrichment of CD63 was observed in both isolated sEV and mEV fractions. CD81 and Alix were predominantly present in sEVs. (B–D) Detection of NME1 and NME2 proteins in cell and EV lysates. (B,D) NME1 protein was present in cell extracts and mEV fractions of (B) FLAG::NME1, and (D) the control vector-transfected cells. (B,D) In sEVs NME1 was not detectable by either antibodies. (C,D) NME2 protein was detected in cell extracts and (C) in both mEV and sEV fractions of the MYC::NME2 expressing cell line by using anti-MYC and NME2 specific antibodies.
Fig 5: Preparation and identification of dECM and characterization of exosomes. A Images of nucleus pulposus tissue decellularization and preparation of hydrogels. B DAPI and H&E staining demonstrated decellularization; Alcian blue staining confirmed the retention of glycosaminoglycan; Sirius red staining confirmed retention of collagen fibers. Scale bar = 20 µm. C Quantitative determination of DNA content and D collagen retention. Data are expressed as the mean ± SD (n = 3). ****P < 0.0001, *P < 0.05. E Cell count in the fluorescence field (n = 3). F Exosome characterization of Alix, TSG101, and calnexin by Western blotting. G TEM analysis of exosomes. Scale bar = 0.1 µm. H NTA analysis of exosomes
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