Fig 1: TGF-ß increases the release of EVs from oral cancer cells. a, b Nanoparticle tracking analysis of EVs from the conditioned medium of HSC-4 cells treated without (a Ctrl-EVs) or with TGF-ß (b Tß-EVs) using the NanoSight system. The inset: NanoSight capture images show particles isolated from the same volume of each conditioned medium. c Immunoblotting analysis of total cell lysate of HSC-4 cells stimulated without (Ctrl) or with 3 ng/ml TGF-ß (TGF-ß) for 72 h, and the respective EV fraction (Ctrl-EVs and Tß-EVs). Samples derived from the same number of particles were loaded onto each lane. Antibodies against EV markers (Alix, TSG101, and CD63) and Golgi apparatus marker (GORASP1) were used for analysis. Cropped blot images are shown. See Supplementary Information for the original full-length blot images. The intensity of each band was then quantified. The relative value was normalized to that of the cell lysate and is shown as ratio
Fig 2: EV characterization from RL cell lines and sera. Nanotrack analysis (Nanosight, a) and TEM (c) for EVs isolated from RL cell line (Lipo246); Nanosight (b), TEM (d) for EVs isolated from serum. WB showing presence of Alix, CD9 and low level/absence of Calnexin in the EVs isolated form cell line (e), WB showing presence of CD9, CD81 and low level of ApoE and Albumin in the EVs isolated from sera (f)
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.
Supplier Page from MilliporeSigma for Anti-ALIX (C-terminal) antibody produced in rabbit