10628D

antibody from Invitrogen Antibodies

Targeting: CD63 ME491, MLA1, TSPAN30

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Product number: 10628D - Provider product page
Provider: Invitrogen Antibodies
Product name: CD63 Monoclonal Antibody (Ts63)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: Tested in western blotting under non-reducing conditions.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: Ts63
Vial size: 200 µL
Concentration: 0.5 mg/mL
Storage: Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C

CD63 protein structure - 10628D shown in red.

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Supportive validation

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Experimental details: Fig. 4 Characteristic of EVs derived from UC-MSCs cultured in xeno-free media (UC-MSC-EVs). a Size analysis of EVs using qNano system (Izon Science Ltd). Representative image is shown. b Western blot analysis of selected proteins in UC-MSC-EVs. Three hundred micrograms of protein extracts was used to detect expression of transmembrane (CD63) and cytosolic (syntenin) proteins. Expression of beta-actin was used as control. c Transcript level for extracellular protein (IL-8) measured by RT-qPCR in UC-MSC-EVs. d Surface antigen profile of UC-MSC-EVs by high-sensitivity flow cytometry. The EV samples were stained with the SYTO(r) RNASelect(tm) Green Fluorescent Cell Stain (Molecular Probes) and selected antibodies labeled with a fluorochrome and further analyzed on an A50-Micro Flow Cytometer (Apogee Flow Systems). The percentage of particles positive for indicated surface marker was analyzed from SYTO(r) RNASelect(tm)-positive objects (in gate R1). Representative dot plots for M1-EVs are shown. e Analysis of transcript levels for genes involved in the maintenance of pluripotency ( NANOG ) or differentiation toward cardiac ( GATA4 ) and endothelial lineage ( FLK1 ) performed with the real time PCR method in UC-MSC-EVs. f Relative transcript levels in EVs compared to parental UC-MSCs. Results are shown as mean +- SD. Results were compared with one-way ANOVA and Dunnet's post hoc test, relative to control conditions ( M6 ). * p < 0.05. UC-MSC umbilical cord-derived mesenchymal

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Experimental details: Figure 1 MDAMB231 breast cancer cells shed exosomes and exosomes and microvesicles (MVs). ( A ) Outline of procedure used to isolate exosomes and MVs from conditioned medium. ( B ) Western blot analysis using CD-63, IkappaBalpha, and flotillin antibodies was performed on lysates of MDAMB231 cells (lane labeled WCL), and the exosomes (lane labeled Exos), and MVs (lane labeled MVs) that these cells generated. A sample containing all extracellular vesicles (EVs) (including both MVs and exosomes) generated by the cells (lane labeled EVs) was also included on the blot. ( C ) Transmission electron microscopy (TEM) image of exosomes isolated from MDAMB231 cells. Scale bar = 50 nm. ( D ) Histogram showing the sizes of exosomes detected in C. ( E , F ) NIH-3T3 fibroblasts were cultured in serum-free media supplemented without (images and bars labeled Serum Starved) or with either 2% serum (images and bars labeled 2% Serum), or 0.5 x 10 6 exosomes/mL collected from MDAMB231 cells (images and bars labeled Exos) for two days, at which point the cells were stained with DAPI to label nuclei. ( E ) Representative fluorescent images of the nuclei from cells cultured under each of the indicated conditions. Asterisks indicate condensed/blebbed nuclei, a hallmark of apoptosis. Scale bar = 10 mum. The boxed portion of the fibroblasts treated with exosomes (Exos) represents an enlarged image and was placed below the other images to further highlight the differences between a normal (non-apoptotic

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Experimental details: Figure 3 Survivin is highly enriched in exosomes from PTX-treated cancer cells. ( A ) Western blot analysis using Survivin, flotillin, IkappaBalpha, and CD-63 antibodies was performed on lysates of MDAMB231 cells treated with either DMSO or PTX (lanes labeled WCL), as well as the exosomes (lanes labeled Exos) and MVs (lanes labeled MVs) generated by the cells. ( B ) The relative amounts of Survivin detected in exosomes generated by DMSO- and PTX-treated MDAMB231 cells. ( C ) Western blot analysis using Survivin and actin antibodies was performed on lysates of MDAMB231 cells treated with PTX for increasing lengths of time. ( D ) Immunofluorescence using a Survivin antibody was performed on MDAMB231 cells treated with either DMSO or PTX (top images). The cells were also stained with DAPI to label nuclei (bottom images). Arrows indicate areas where Survivin is detected as puncta in the cytosol of cells treated with PTX. Scale bar = 5 um. ( E ) Western blot analysis was performed using a Survivin antibody on lysates of MDAMB231 cells, U87 glioblastoma cells, and SKBR3 cells that had been treated with DMSO or PTX (lanes labeled WCL), and on the exosomes these cells generated (lanes labeled Exos). ( F ) Western blot analysis was performed on lysates of exosomes from MDAMB231 cells that had been treated with the indicated chemotherapeutic agents and inhibitors. The experiments in B were performed a minimum of three separate times, with each experiment yielding similar results. Stude

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Experimental details: Figure 6 Characterization of exosomes derived from CL 187 cells and CRC patient plasma. A, Representative electron microscope images of exosomes isolated from CL 187 cells. B, Western blotting analysis of LEA and exosome-marker expression in exosomes from CL 187 cells using ND -1, anti- TSG 101, and anti- CD 63, respectively. C, Representative analysis of LEA expression in exosomes from CRC patient and healthy donor plasma by western blotting analysis using ND -1

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Experimental details: Figure 1 Isolation and characterization of exosomes. (A) Image of exosomes observed by transmission electron microscopy. (B) Size distribution of exosomes secreted by non-treated HCT116 cells. (C) Western blot of alpha-tubulin, CD63 and CD81 in exosomes and cells.

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Experimental details: Fig 2 Characteristics of exosome-depleted FBS. In (a) Quantification of exosomes present in the cell culture medium supplemented with STD FBS or ED FBS at the input and in the post-UC fractions (supernatant and pellet) based on ACHE activity. In (b) Western blot of CD63 in the cell culture medium either before (input) or after UC (supernatant); Ponceau S staining demonstrates the serum albumin content. In (c) Coomassie blue staining of electrophoretically-separated proteins from standard (STD) and exosome-depleted (ED) FBS. In (d) Effects of supplementation with FBS on cell viability; FaDu cells were cultured for 24 h in a medium supplemented with STD FBS, ultracentrifuged STD FBS (STD-UC) or ED FBS.

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Experimental details: Fig 3 Exosome isolation by the size exclusion chromatography. In (a) representative immunoblot showing the distribution of exosome markers (CD63, CD9, CD81) and high-abundance serum proteins (illustrated by Ponceau S staining) in the successive SEC fractions of a FaDU culture medium. In (b) total protein concentrations (mug/uL) in the subsequent SEC fractions. In (c) culture medium supplemented with 5% ED FBS and NOT co-cultured with cells was analyzed as in Panel A; ""E+"" denotes exosome-containing positive control.

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Experimental details: Figure 1 Generation and characterisation of TRAIL-loaded EVs (TRAIL-EVs). ( A ) Flow cytometry analysis of TRAIL expression in 293T cells transfected by empty virus (293T) or TRAIL-expressing lentiviruses (293TflT); ( B ) Immunofluorescent detection of TRAIL expression (red) by a PE-anti human TRAIL antibody in 293T and 293flT cells with nuclei labeled as blue by DAPI; ( C ) Electron microscopy of EVs isolated and purified by ultra-centrifugation of 0.22 um filtering cleared supernatant of 293TflT cells grown in EV-depleted FBS containing medium; ( D ) Assessment of size distribution of isolated EVs by a Flow Nanoanalyzer that can detect nanoparticles with size ranging from 7 to 1000 nm; ( E ) Measurement of TRAIL expression levels in EVs derived from 293T (293T-EV) or 293TflT(293TflT-EV) using a commercial TRAIL-specific ELISA kit. Values are mean +- S.E.M ( n = 3). ( F ) Western blotting detection of TRAIL, tetraspanin CD63 and loading control protein alpha-tubulin in EVs or cellular lysates; for each sample 10 µg of cellular or EV proteins were analyzed.

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Experimental details: Fig. 3 Schema summarizing the protocols used to extract muscle exosomes from the primary human myotube culture medium, using either the ultracentrifugation or the modified polymer-precipitation strategy. For a single data point, 14 flasks of 225 cm 2 are plated with 7.5 x 10 6 myoblasts. After 24 h, once the myoblasts have attached to the flask, they are rinsed 6 times in DMEM and then differentiated into myotubes by cultivating them in DMEM. After 72 hr, the conditioned medium is collected for muscle exosome extraction. After removing dead cells (200 g, 10 min, RT), cell debris (4000 g, 20 min, 4 degC), and ectosomes (20,000 g, 70 min at 4 degC, and filtered at 0.22 mum), the cleared media is subjected to exosome extraction either by the ultracentrifugation protocol or by a modified polymer-precipitation protocol. Ultracentrifugation is at 100,000 g (70 min, 4degC), which is followed by washing the pellets three times with PBS (100,000 g, 70 min, 4degC). The subsequent pellet is then either resuspended in 100 mul of PBS or in NuPAGE(tm) LDS sample buffer for western blot. For the modified polymer-precipitation protocol, the polymer is added at half the volume of the pre-cleared media and incubated overnight at 4 degC. The mix is then centrifuged at 10,000 g for 70 min at 4 degC. The subsequent pellet is then washed 3 times in PBS using a 100 kDa Amicon(r) filter column. Western blot shows the rescue of the epitope CD63 after 3 washes in PBS

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Experimental details: Fig. 4 Validation of exosome extraction strategy . For each experiment, exosomes were extracted from the culture medium of 100 x 10 6 myoblasts differentiated into myotubes for 3 days using either the ultracentrifugation or the polymer precipitation. The culture medium was non-supplemented DMEM (without serum). a Cup-shaped vesicles were observed by electron microscopy with both extraction protocols. bar = 100 nm. b Both extractions show vesicles that are positive for CD63 and CD82 by electron microscopy. Bar = 100 nm. c Exosome extracts were loaded on iodixanol gradients as described in material and methods. Western blot results are shown for CD63 and CD81 in twelve fractions for the iodixanol gradient. Top panel: exosomes extracted by ultracentrifugation. Bottom panel: exosomes extracted by polymer-based precipitation. Exosomes were detected at a density of 1.15-1.27 g.ml -1 . d Nanosight analyses show similar-sized vesicles using both strategies, from 100-200 nm, with a greater number of particles being detected when using the polymer extraction. e Proteomic analysis of muscle exosomes. Venn diagram showing the overlap between muscle exosomes and proteins known to be detected by mass spectrometry in exosomes (Vesiclepedia, Exocarta database [-])

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Experimental details: Fig. 5 Polymer-based precipitation efficiently extracts functional exosomes from 7.5 x 106 cells. a SDS-page protein quantification showing that the greatest efficiency in terms of exosomal protein per cell plated was obtained when exosomes were extracted from differentiated myoblasts at a density of 33,400 cells.cm -2 . Differentiated myoblasts were plated at 14,147 (lane 1), 33,400 (lane 2), or 106,100 (lane 3) cells per cm 2 . Right panel: representative SDS-page stained with Coomassie. Left panel: protein concentration measurements in secreted exosomes from cells plated at a different density. *, ***, P < 0.05 and P < 0.001, significantly different from 33,400 and 106,100 cells.cm 2 . ( n = 4, 3, 4 per condition). b Muscle exosomes were positive for CD63, CD81, Flotillin, HSPA8, and Alix. c mRNA was detectable with a clean profile from polymer-precipitated exosomes of 7.5 x 10 6 differentiated myoblasts. No 18 s and 28 s RNA were detected, indicating that there were no RNA contaminants from dead cells. Inset panel: Representative electrophoresis obtain with Agilent 2100 Bioanalyzer for myotubes and exosome RNA extract. d Western blot showing that polymer precipitated exosomes from 7.5 x 10 6 differentiated myoblasts can be used to pull down a specific subpopulation such as CD63 positive exosomes (+/-CD63 = with/without anti-CD63 antibody). e Polymer-precipitated exosomes (pre-stained with PKH26 following extraction; red channel) were capable of integrating into myotubes o

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Experimental details: FIGURE 4 Positive detection for specific exosomal surface markers (CD 63, CD9, and CD81) and for the control-positive marker (b-actin) by immunoblotting. (A) exosomes isolated by UC and (B) TEIp kit.

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Experimental details: Fig 4 Protein markers of MDM-derived sEVs. (A) Representative image of three independent assays of polyacrylamide gel stained with silver nitrate after separation of 40 mug total protein from cell (Cell) or sEVs pool lysates (EVs; pools comprise samples from four individual donors). (B) Western blot analysis of sEVs markers (Alix, CD63, and CD81) and non-EVs markers (Calnexin and Cytochrome C) ( n = 3). beta-actin = loading control. 40 mug of total protein were loaded onto the gel. MW: molecular weight marker.

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Experimental details: Figure 2 Successful isolation of exosomes from blood plasma was verified by Transmission Electron Microscopy (TEM), Western Blot, and Nanoparticle tracking. ( A ) Two representative TEM graphs showing negatively stained exosomes isolated from an HNSCC patient. As indicated by the size bars, exosomes vary in diameter between 30 and 150 nm and have round to oval shapes. Size bar on the top TEM graph = 500 nm, size bar on the bottom TEM graph = 200 nm. ( B ) Western Blot analysis of exosomes was performed to confirm the expression of exosomal markers TSG101, CD9 and CD63 and the expression of epithelial cell marker EpCAM (upper frame). Exosomes were also analyzed for negative markers ApoA1 and Grp94 along with plasma (diluted 50x in PBS) and cell lysate samples as positive controls. MW marker, positive control molecular weight marker. ( C ) Size distribution of exosomes was measured by nanoparticle tracking. The mean diameter was 86.8 nm. The maximal and minimal diameters were 257.5 nm and 22.5 nm, respectively. The 90th and 10th percentile were at 121.2 and 53.6 nm, respectively. ( D ) Protein content of exosomes was determined by Bicinchoninic Acid (BCA) Assay. Average protein content: 80.9 g/mL (HNSCC exosomes), 69.2 g/mL (healthy volunteer exosomes). n = 23 (HNSCC), n = 10 (NC). HNSCC, exosomes from blood plasma of HNSCC patients. NC = no cancer, exosomes from blood plasma of healthy volunteers. ( E ) B cells that were not co-cultured with exosomes exhibited colony formation

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Experimental details: FIGURE 2 Total IRS-1, p-IRS-1 and downstream substrates level in blood neuron-derived extracellular vesicles. (A) The representative protein blot images of IRS-1 and different phosphorylated forms of IRS-1, including Y612, S616, S636/639, and S312. HSP70 and CD63 were exosomal proteins and markers. GAPDH was the protein loading control (1 and 2 indicate different samples). Comparison of the levels of total IRS-1, p-IRS-1 Y612 , p-IRS-1 S616 , p-IRS-1 S636/639 , p-IRS-1 S312 (B) , and downstream IRS-1 substrates (C-E) between healthy controls [with or without diabetes mellitus (DM)] and Parkinson's disease (PD) patients (with or without DM). The phosphorylation status of IRS-1, P70S6K, Akt, and Erk was normalized to total form IRS-1, P70S6K, Akt, and Erk. (F) The EV markers-CD63 and HSP70 was not different between 4 groups. Data was presented as mean +- SEM. * p < 0.05.

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Experimental details: Figure 1 Human BMSC-derived sEVs can be taken by MM cells. (A) Transmission electron microscopy images of human BMSC-derived sEVs. (B) Size distribution of human BMSC-derived sEVs was determined using the nanoparticle tracking analysis. (C) Exosomal positive markers, including CD63, CD9, flotillin-1, as well as a negative marker calreticulin, in BMSC and BMSC-derived sEV lysate were measured using western blot. (D) 50 mug/mL DID-labeled BMSC-derived sEVs or DID control solution was added to four MM cell lines, including RPMI 8226, MM1S, U266, and H929. After 24 h of culture, the fluorescence signal of DID in these cells was examined using flow cytometry. PBS was added to cells and included as a control. (E) 50 mug/mL DID-labeled BMSC-derived sEVs were added to four MM cell lines and the mean and median fluorescence intensity of DID in these cells was determined using flow cytometry after the culture for indicated times. n=3. Error bar, mean +- SD.

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Experimental details: FIGURE 6 Purity of EV samples assessed by particle/protein ratio and Western blotting. (a)-(c) The protein concentrations of EV preparations separated by different methods from CCM, urine and plasma. The protein concentrations have been corrected for initial sample input volume. (d)-(f) The particle/protein ratios of EV preparations separated by different methods from CCM, urine and plasma. The particle numbers were measured by nFCM. The error bars represented the standard deviation of three repetitive experiments. * P < 0.05, ** P < 0.01, one-way ANOVA analysis with Tukey multiple comparison test as well as a variance-covariance model. (g) Western blots of flotillin-1, CD63, CD81, and calnexin for EV preparations separated from CCM by different methods. 9 mug of protein from each EV preparation was loaded. (h) Western blots of flotillin-1, CD63, CD81, THP and calnexin for EV preparations separated from urine by different methods. 3.3 mug of protein from each EV preparation was loaded. (i) Western blots of flotillin-1, CD81, APOA1 and calnexin for EV preparations separated from plasma by different methods. 25 mug of protein from each EV preparation was loaded. MCF7 membrane and cytosolic protein fractions served as positive and negative controls for flotillin-1, CD63, CD81 and calnexin. Whole urine sample was used as positive control and MCF7 cellular fractions as negative control for THP. Recombinant human Apolipoprotein A1 served as positive control for APOA1.GAPDH staining

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Experimental details: Fig. 1 Characterization of extracellular vesicles (EVs) released by HaCaT cells. a Western blotting analysis of purified vesicles (EV) for exosomal surface markers (CD9, CD63) and endoplasmic reticulum marker (GRP94) side by side with HaCaT cell lysate (CL). b Venn diagram of identified proteins and proteins in the vesiclepedia database. The pink area represents proteins from the vesiclepedia database and the blue area represents proteins we identified. The identified proteins in HaCaT cell-derived EVs were analyzed using FunRich Bioinformatic Resources and classified by c biological process, d cellular component, or e molecular function

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Experimental details: Figure 1 Characterization of exosomes isolated from plasma. ( A ) Representative transmission electron microscopy (TEM) image of exosomes. Scalebar = 200 nm. ( B ) Representative size distribution of exosomes measured by nanoparticle tracking analysis (NTA). ( C ) Exosomes derived from plasma of healthy donors (HD) and head and neck squamous cell carcinoma (HNSCC) patients were analyzed by Western blot for the presence of exosome specific markers using antibodies against CD63 and CD81 under non-reducing conditions and antibodies against CD9 and TSG101 under reducing conditions. Western blot analysis for the negative marker Grp94 and the apolipoprotein Apo1A was also performed for exosomes, cells, and plasma.

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Experimental details: Figure 2 Successful isolation and characterization of engineered EVs displaying peptides of interest. (A) Representative size distribution of the naive, pep1- and p88-display EVs determined by Nanoparticle Tracking Analysis. (B) Transmission electron microscopy images of naive, pep1-, and p88-EVs showing gold labeled HA and CD63 surface markers. (C) Western blot analysis of engineered EVs (p88 fusion peptide-44Kda; pep1 fusion peptide-42KDa) for the presence of EV biomarkers CD63(30-60KDa) and TSG101(44KDa), and peptide HA-tag. Additionally, analysis of cell lysate and engineered EVs for cellular biomarkers Calnexin (67KDa) and beta-actin (42KDa) (D) Summary of particle number and pDNA copy numbers determined by NTA and qPCR of pep1- and p88-display EVs. (E) pDNA quantification before and after DNase I treatment of EVs. Naive EVs mixed with pDNA and p88 EVs were treated with DNase I. pDNA were quantified by qPCR following pDNA isolation. *pDNA undetected

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Experimental details: Fig. 3 Characterization of control EVs and Cur-EVs. a Representative western blot image showing bands of standard surface markers (CD9, CD63, CD81) of control EV- and Cur-EV lysates. b , c Morphology of control EVs and Cur-EVs was monitored by TEM; scale bar, 100 nm. d , e Particle size distribution of control EVs and Cur-EVs was measured by NTA. f , g Quantitative comparison between control EVs and Cur-EVs in count and size measured by NTA; n = 3. h Absorbance at 420 nm of control EVs and Cur-EVs was determined with by spectrophotometry indicating presence of curcumin in Cur-EVs; n = 3. i Cell nuclei were stained with DAPI (blue) and chondrocytes were stained with Phalloidin (green) to visualize the structure of the cytoskeleton. PKH26-labeled control EVs (red) and Cur-EVs (red) were internalized by chondrocytes and visualized with fluorescent microscopy

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Experimental details: Figure 5. Release of exosomes from Mtb H37Rv-infected or naive THP-1 cells. THP-1 macrophages were infected with Mtb H37Rv (Mtb + ) for 4 h (MOI 5) in DCM-UG medium for 4, 24 and 48 h at 37degC and in the presence of 5% CO 2 . Exosomes were extracted from cell culture supernatants using total exosome isolation reagent and were further analyzed by western blot analysis for the exosomal protein markers, (A) CD63, CD81, CD9 and (B) LAMP-1, which served as a positive control for exosomes signal and Mtb proteins [Ag85 and Mpt64 and recombinant Mtb Mpt64 protein (His-tag)]. Data from one representative experiment out of at least three independent experiments are shown. MOI, multiplicity of infection; Mtb, Mycobacterium tuberculosis ; DCM-UG, ultra-centrifuged CellGenix (r) GMP DC Medium; LAMP-1, lysosomal associated membrane protein-1; Ag85, mycobacterial antigen 85.

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Experimental details: 7 Figure Effect of RAB5A knockdown on exosome secretion. (A), Quantification of total protein of exosome samples isolated from RAB5A knockdown and scrambled cells ( n = 3, two-tailed Student's t test, mean +- SEM ). (B), Representative image of western blot of exosomal markers, CD63 and Alix. The total protein of exosome samples were loaded based on an equal number of cells (5 x 10 6 ), whereas an equal amount of whole-cell lysate (25 ug) was loaded. One experiment out of three is shown. (C), Quantitative analysis of CD63 and Alix expression in exosome samples isolated from RAB5A knockdown cells compared with scrambled cells. The red horizontal dashed line indicates the level of scrambled cells as control (CD63 n = 4, Alix n = 3, two-tailed Student's t test, mean +- SEM ). sh-Ctrl, stable scrambled cells; sh- RAB5A KD, stable RAB5A knockdown cells; * p

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Experimental details: Fig. 2 Effect of deuterium labelling on cell viability and extracellular vesicle production. (a-c) Effect of deuterium labelling on (a) cell viability ( N = 2, n = 12), (b) hydrodynamic diameter of isolated EVs ( N = 6, n = 3), and (c) particle number of isolated EVs ( N = 6, n = 3) for MDA-MB-231 cells cultured on deuterium-containing medium (containing D 2 O, d-Chol, or d-Gluc) for 72 hours as compared to untreated controls, measured via nanoparticle tracking analysis. Data presented as mean SD +- (one-way ANOVA, Tukey's honest significant differences post hoc correction, * P < 0.05, ** P < 0.01, *** P < 0.001). (d-f) Expression of three different EV markers, (d) CD9, (e) CD63, and (f) CD81, for EVs isolated from MDA-MB-231 cells cultured on deuterium-containing medium (containing D 2 O, d-Chol, or d-Gluc) for 72 hours as compared to untreated controls measured via immunoblotting analysis ( N = 6, n = 1). Data presented as mean +- SD.

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Experimental details: Figure 1 Protein sample were profiled with TotalSeq antibody. ( a ) Basic work flow for detection of immobilized protein sample by TotalSeq-A antibody. Structure of conjugated oligo in TotalSeq-A antibody (up right), reprint from BioLegend website with permission. Size and comparison of pre-cut strip and PCR tube (bottom right). ( b ) Melting curve of different TotalSeq antibodies on EV protein samples in triplicate. ( c ) Intact (upper left) or lysised (upper right) #1 EV samples were tested with TotalSeq CD63 (green) and control mIgG (blue) antibody in Triplicate. Equal amount of BSA (triangle) was used as negative control. Ct values of CD63 (white) and control mouse IgG (gray) are overlaid in column chart to show the difference. ( d ) Serial dilution of #1 EV sample was incubated with TotalSeq antibody CD63 (square), CD81 (triangle), EGFR (diamond) and control IgG (circle). Amplification curve (bottom) of CD63 on EV dilution (green) and BSA (blue). All experiments were performed in duplicate. ( e ) CD63 immunoblot result of EV and whole cell lysate. Full-length blots are presented in Supplementary Figure S7 . ( f ) TotalSeq qPCR result on the same samples as in ( e ). Data were normalized to wild type HEK293 samples.

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Experimental details: Fig. 1 Preparation and identification of ICG/PTX@RGE-EV. A Morphological characteristics of EVs derived from Raw264.7 cells under TEM (scale bar = 100 nm). B Diameter of EVs detected by NTA. C Protein expression of EV markers, CD63, CD81, Alix and EV contamination index ApoA1 detected by Western blot analysis. D Morphological characteristics of ICG/PTX@RGE-EV under TEM (scale bar = 100 nm). E Diameter of EVs detected by NTA. F Protein expression of EV markers, CD63, CD81, Alix and EV contamination index ApoA1 in ICG/PTX@RGE-EV detected by Western blot analysis. G ICG/PTX@RGE-EV observed under super-resolution fluorescence microscopy (green fluorescence: RGE) (scale bar = 20 mum). H ICG/PTX@RGE-EV under confocal microscope (red fluorescence: EV, green fluorescence: RGE) (scale bar = 20 mum). I U251 cells incubated with CM-DiI-labeled ICG/PTX@RGE-EV observed under confocal microscope (scale bar = 20 mum). J FITC-positive cells identified by flow cytometry (i, U251 cells incubated with ICG/PTX@RGE-EV; ii, U251 cells incubated with ICG/PTX@ EV and RGE; iii, U251 cells incubated with ICG/PTX@EV; iv, free U251 cells; v, FITC-labeled U251 cells). Measurement data were expressed as mean +- standard deviation. Data comparison was analyzed by one-way ANOVA among multiple groups, followed by Tukey's post hoc test. * p < 0.05 vs. the i group. The experiment was repeated in triplicate

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Experimental details: Additional file 1: Fig. S1. Representative images for Western blot analysis of EV markers CD63, CD81, Alix and EV contamination index ApoA1.

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Experimental details: Additional file 5: Fig. S5. In vitro stability of ICG/PTX@RGE-EV. A, Concentration of RGE-peptide bound on EVs. B, ICG concentration in EVs. C, PTX concentration in EVs. D, Expression of EV markers CD63, CD81 and EV contamination index ApoA1 in ICG/PTX@RGE-EV and Raw264.7 cells detected by Western blot analysis. E, Diameter and Zeta potential of EVs detected by NTA. Measurement data were expressed as mean +- standard deviation. Data comparison was analyzed by one-way ANOVA among multiple groups, followed by Tukey's post hoc test. The experiment was repeated three times independently.

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Experimental details: FIGURE 2 Characterization of control EVs and gene analysis of hsa-124-3p in serum-derived EVs and serum. (A) Representative image of Western blotting showed bands of standard surface markers ( CD9 , CD63 , and CD81 ) of EVs and EV-depleted serum. (B) Morphology of EVs was monitored by TEM; scale bar: 100 nm. (C,D) Particle size distribution of EVs was determined by NTA. (E,F) Gene expression of hsa-miR-124-3p in EVs and serum of different samples at different time points. Compared to the control group: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, # Difference between groups: # p < 0.05; ## p < 0.01; #### p < 0.0001; one-way ANOVA with the Newman-Keuls multiple comparison test.

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Experimental details: Fig. 2 Protein analyses of kidney EVs by western blot and mass spectrometry. a Western blots of lysed kidney EVs demonstrate presence of flotillin-1, CD63, and CD81 and absence of cellular debris marker calnexin. Control samples were MCF7 membrane (HI) and cytosolic protein (LO) fractions. b Bubble plot of EV protein expression across kidney EVs demonstrates a similar pattern of EV protein expression as seen on the western blots. For each marker, log 2 ratios were calculated by normalization to the reference channel (pooled cellular reference). c Functional Gene Ontology (GO) annotation of the EV-associated proteins commonly identified across six renal cell lines

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Experimental details: Figure 6 Tumor cell-derived extracellular vesicles are enriched in manganese and affect tumor cell migration. LLC cells-derived extracellular vesicles were concentrated by ultracentrifugation. ( a ) EVs quantification and size determination. N = 6. ( b ) CD63 and syntenin-1 detection by Western blotting. Ponceau was used as total protein control. ( c ) Tumor cells, EV-enriched fraction, EV-free conditioned medium and basal medium were analyzed by X-Ray Fluorescence. N = 6. Units are expressed in concentration as ppm (parts per million). Control cells--white bars; Mn-pretreated cells (Mn) - black bars. *p < 0.05, Student's T test. ( d ) Wound healing assays of LLC cells incubated with extracellular vesicle-enriched medium from control and Mn-pretreated LLC cells. N = 12. *p < 0.05; **p < 0.01, one-way ANOVA and Bonferroni's Multiple Comparison Test.

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Experimental details: FIGURE 1 VE characterization from explant culture supernatant. (A) Representative vesicle size distribution (nm) by NTA of VE. (B) Western blot analysis for the CD63 exosome marker. (C) Quantification of VE by ELISA measuring CD63 (controls vs. EO-PE p = 0,2069; controls vs. LO-PE p = 0,0262; EO-PE vs. LO-PE: p = 0,0175). (D) Quantification of protein within VE by Bradford method (controls vs. EO-PE p = 0,2069; controls vs. LO-PE p = 0,6031; EO-PE vs. LO-PE: p = 0,3129). (E) Analysis of ratio of protein quantity by VE (controls vs. EO-PE p = 0,0175; controls vs. LO-PE p = 0,3759; EO-PE vs. LO-PE: p = 0,0262) * p

Supportive validation

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Experimental details: Figure 3 Characterization of OOM-SC-EVs. ( A ) TEM images of EVs showing cup- or sphere-shaped morphology. TEM analysis was performed at 80 kV. Scale bar = 100 nm. ( B ) Characterization of the expression levels of the EV-associated positive surface markers, including CD9, CD63, CD81, calnexin, and GM130, via immunoblotting. We used 3 ug of EVs for the immunoblotting analysis. The protein expression levels of the EV markers were compared with those of the whole cell lysate. ( C ) Dynamic light scattering analysis of EVs sizes. The average diameter of the purified EVs was of a size range of 90 to 120 nm. ( D ) Nanoparticle tracking analyzer NS300 for counting EV numbers. EVs concentrations were 1.66 x 10 10 particles/mL for OOM-SC-EVs and 1.5 x 10 10 particles/mL for WJ-MSC-EVs. ( E ) FACS analysis confirmed the expression of EV-associated markers, namely CD63 and CD81. ( F ) Phase-contrast microscopic pictures of WJ-MSCs monolayer and spheroids of WJ-MSCs and OOM-SCs (OOM-SC-74). For spheroid culture, WJ-MSCs and OOM-SCs were seeded onto F127 -coated AggreWell 400 TM plates for 24 h for EB formation. Then, EBs were transferred to the ORS at 60 rpm for two days. EVs were isolated from both spheroids and the monolayer simultaneously. Scale bar = 200 mum. ( G , H ) Nanoparticle tracking analyzer NS300 for counting EV numbers in monolayer and spheroids. We detected 1246 particles/cell in WJ-MSCs monolayer, whereas EVs yield from WJ-MSCs and OOM-SCs spheroids were 3984 particles/c

Supportive validation

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Experimental details: Figure 1 Characterization of small extracellular vesicles (sEVs) derived from human Head and Neck Squamous Cell Carcinoma cell lines: FaDu (hypopharyngeal squamous cell carcinoma), PCI-30 (tongue squamous cell carcinoma), SCC-25 (tongue squamous cell carcinoma); and sEVs derived from HaCat (immortalized human keratinocytes). ( A ) Representative cryogenic transmission microscopy image of HNSCC cell-derived sEVs. ( B ) Representative concentration and size distribution plot of HNSCC-derived sEVs measured by nanoparticle tracking analysis (NTA) and particle visualization based on Brownian motions. ( C ) Immunoblotting of sEV markers CD63, CD9, and negative marker Grp94 in HNSCC-derived sEVs. Full blot images are presented in Figures S2-S4 . ( D ) Particle concentration related to normoxic (21% O 2 ) and hypoxic (1% O 2 , 5% O 2 , 10% O 2 ) conditions. Results were obtained using NTA and normalized to the protein levels in lysates of producer cells. ( E ) Particle diameter related to normoxic (21% O 2 ) and hypoxic (1% O 2 , 5% O 2 , 10% O 2 ) conditions. Results were obtained using NTA. All data represent three biological replicates and are presented as means +- SD. * p < 0.05 vs. 21%; ** p < 0.01 vs. 21%; *** p < 0.001 vs. 21%.

Supportive validation

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Experimental details: Figure 1 Characterization of control EVs and HR-EVs. EVs were processed for western blot, TEM and NTA after isolated from 500mul serum of control and HR groups. (A) The same amount (30mul) of control EVs, HR-EVs and EVs-depleted-Serum were identified using western blot. Representative western blot image showing bands of standard surface markers (CD9, CD63, CD81) of control EVs and HR-EVs. (B, C) Morphology of control EVs and HR-EVs was monitored by TEM; Scale bar: 100 nm. (D, E) Particle size distribution of control EVs and HR-EVs was determined by NTA. Distribution of EVs with a size of 90-200 nm in diameter in both groups. (F, G) Quantitative comparison between control EVs and HR-EVs in count and size measured by NTA; n = 10; All values represent mean +- standard deviation. * p < 0,05, independent two-tailed Student's t -tests.

Supportive validation

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Experimental details: Figure 2 HR promoted the concentration of EVs and CD203c + -EVs and CD63 + -EVs derived from serum. 500mul serum from control and different time point HR groups were used to pellet EVs, finally, the EVs were resuspended in 200mul PBS. The total concentration of EVs was determined by a BCA assay. The protein expression levels of CD63, CD203c, CD9 and CD81 in EVs/serum were identified using western blotting. (A) Total concentration of EVs isolated from same volume serum of control and different time point HR groups, n = 10. (B-F) Relative CD63, CD203c, CD9 and CD81 protein expression levels of EVs derived from same volume of serum in control and HR groups, 30mul EVs/lane, n = 5 (G-I) Relative CD63 and CD203c expression levels in same volume of serum in the control and HR groups, 30mul serum/lane, n = 5. All values represent the mean +- standard deviation. Difference to control: ** p < 0,01; *** p < 0,001; # Difference between groups: # p < 0,05; ## p < 0,01; 1 way ANOVA with Newman-Keuls Multiple Comparison Test.

Supportive validation

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Experimental details: FIGURE 6 Characterization of plasma exosomes and uptake of exosomes by macrophages. (A) Electron microscopy observation of whole-mounted exosomes purified from plasma. (B) Average overall size distribution of exosomes from plasma using nanoparticle tracking analysis. (C) The proteins from isolated exosomes were initially characterized on Western blot to assess the expression of CD63 and CD9. (D) Internalization of PKH67 (green)-labeled exosomes in THP1 cells induced by phorbol-12-myristate-13-acetate was captured by living cell imaging. Fluorescent cellular imaging was carried out using the green fluorescent protein channel for PKH67 fluorescence-labeled exosomes (green). Cell counts were detected with bright field. (E) The relative object integral fluorescence was measured over time with the uptake of exosomes. (F) The percentage of uptake rate was calculated with the software.

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Experimental details: Figure 1 Characterization of OCEXs. (A) The sizes of the WSU-HN4- and SCC-9-derived exosomes were determined by NTA. (B) Representative images of exosomes derived from WSU-HN4 and SCC-9 cells, as detected by TEM. Scale bars, 50 nm. (C) Expression of the exosomal markers, Alix, CD63, CD9 and Rab5, in WSU-HN4- and SCC-9-derived exosomes was determined by western blot analysis. (D) Co-expressed proteins in WSU-HN4- and SCC-9-derived exosomes were detected by mass spectrometry. (E) Overlap of OCEX proteins and the top 100 frequently identified exosomal protein markers from the ExoCarta database. OCEXs, oral cancer-derived exosomes; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; EXO/Exo, exosomes.

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Experimental details: Figure 2 Characterization of hBMSC-derived EVs. (A) Representative western blot image ( n = 4) demonstrates standard surface markers (CD9, CD63, and CD81) of hBMSC-derived EVs (lane 1: hBMSC lysate; lane 2: hBMSC-EVs). (B) Particle size distribution of hBMSC-EVs was measured by NTA. (C) Morphology of hBMSC-EVs was monitored by SEM, scale bar: 1 mum. (D) Cell nuclei were stained with DAPI (blue) and chondrocytes were stained with phalloidin (green) to visualize the cytoskeleton. PKH26-labeled hBMSC-derived EVs (red) internalized by chondrocytes were visualized with fluorescent microscopy.

Supportive validation

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Experimental details: Figure 3 Characterization of EV-depleted FCS (FCS depl-uc ). Prior to ultracentrifugation, normal FCS was diluted in culture medium to different concentrations (20, 50%, and undiluted = 100%); different FCS depl-uc groups were identified after ultracentrifugation. (A,B) FCS depl-uc-20% was controlled for EV surface makers (CD9, CD63, and CD81) detected by western blotting (lane 1: hBMSC lysate; lane 2: hBMSC-EVs; lane 3: undepleted FCS; lane 4: FCS depl-uc-20% ). Representative western blot image (A) and Ponceau Red-stained images for each surface marker (B) are shown; n = 3. (C,D) Proliferation and apoptosis of hBMSC were determined by BrdU assay and caspase-3/7 activity assay separately after being incubated in culture medium supplemented with the different FCS groups for 24 h. All values represent mean +- standard deviation. *Significant difference to control: * p < 0.05; ** p < 0.01; # Significant difference between groups: # p < 0.05; ### p < 0.001 one-way ANOVA with Newman-Keuls Multiple Comparison Test; n = 4.

Supportive validation

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Experimental details: Fig. 3 Plasma cell exosomal miR-330-3p could be transferred to ovarian cancer. ( A ) Schematic diagram of plasma cells and ovarian cancer cells cocultured in six-well plates. ( B ) Ovarian cancer cells were cocultured in the absence or presence of primary PKH67-labeled plasma cells (green). Nuclei were stained with DAPI (blue) ( n = 3 for each group). Scale bar, 20 mum. ( C ) Western blot analysis for CD63, CD81, and beta-actin in plasma cell exosomes ( n = 3 for each group). ( D ) Electron micrograph of plasma cell exosomes shows the morphological size (50 to 200 nm). Scale bar, 100 nm. ( E ) Size distribution of exosomes was measured using NanoSight analysis. ( F and G ) Immunofluorescent images of PKH67 abrogation in ovarian cancer cells with respective treatment. Scale bar, 20 mum. Statistical chart was plotted ( n = 3 for each group). ( H ) Scheme chart for small RNA sequencing in plasma cell exosomes in patients with ovarian cancer. tRNA, transfer RNA; rRNA, ribosomal RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; piRNA, Piwi-interacting RNA. ( I ) Venn diagram for overlapped miRNAs identified in ovarian cancer plasma cell exosomes. ( J ) Heatmap for unsupervised hierarchical clustering of GSE73582 dataset using plasma cell exosome-specific miRNA panel as classifiers. ( K ) Cellular programs enriched by GSEA for plasma cell exosome-specific miRNAs represented using Enrichment Map. ( L ) The univariate regression analyses of the identified top miRNAs associa

Supportive validation

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Experimental details: Fig. 5 MAAP promotes association of AAV with EVs. A Schematic of EV isolation by iodixanol density gradient from HEK293 suspension culture. B Immunoblots of iodixanol fractions from suspension cells producing recombinant MAAP8Delta vector complemented in trans with CMV-HA and C CMV-MAAP8-HA. EVs, capsid and MAAP were analyzed from the media of HEK293 producing cells at day 3 post transfection. Capsid and MAAP proteins were analyzed by SDS-PAGE under reducing conditions while EV markers (CD81, CD63, CD9) were analyzed by SDS-PAGE under non-reducing conditions ( n = 2). D Graph displaying percent vector genome titer relative to total viral genomes for each fraction from iodixanol gradient purified MAAP8Delta vector complemented in trans with CMV-HA and CMV-MAAP8-HA. E Schematic of EV isolation by size exclusion chromatography (SEC) from HEK293 suspension culture. F Immunoblots of SEC fractions from suspension cells producing recombinant MAAP8Delta vector complemented in trans with CMV-HA and G CMV-MAAP8-HA. EVs and MAAP were analyzed from the media of HEK293 producing cells at day 3 post transfection. MAAP protein was analyzed by SDS-PAGE under reducing conditions while the EV marker CD63 was analyzed by SDS-PAGE under non-reducing conditions ( n = 2). H Graph displaying percent vector genome titer relative to total viral genomes for each fraction from SEC purified MAAP8Delta vector complemented in trans with CMV-HA and CMV-MAAP8-HA.

Supportive validation

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Experimental details: Fig. 1 CD63-positive ferroptosis-dependent EVs (FedEVs) derived from macrophages simultaneously exposed to asbestos and iron in vivo and in vitro . (a) Histological images of peritoneal wall in situ in the murine model 6 months after ip injection of 3 mg crocidolite; hematoxylin and eosin (HE) image (left upper), Berlin blue staining (insoluble iron as bluish green, left lower), phase-contrast with red fluorescent image (right panels); cell-specific marker: MSLN (mesothelin), mesothelial cell; alphaSMA (smooth muscle actin), myofibroblast; CD68, macrophage; white arrowheads, crocidolite (bar = 100 mum). (b) High-resolution fluorescent image in granuloma; red, cell specific marker; green, CD63; blue, Hoechst33342 nuclear staining (bar = 20 mum). (c) Quantification of fluorescent intensity of CD63 in different cells surrounding granuloma (n = 30; mean +- SEM). (d) Schematic image of peritoneal wall surrounding the asbestos-induced granuloma. (e, g) Immunoblot analysis of CD63 in cells under ferroptosis and their media; HT1080 fibrosarcoma cells were exposed to RSL3 (10 muM, 24 h), and phorbol myristate acetate (PMA)-primed THP1 macrophage cells were exposed to crocidolite (15 mug/cm 2 ) in the presence of absence of ferric ammonium citrate (Fe, 100 mug/ml) up to 96 h. (f, h) Membrane-catch transmission electronmicroscopy (TEM) images of EVs on carbon grids (bar = 200 nm). (i-l) The precision of nanoparticle tracking analysis (NTA) concentration measurements counted size-distrib

Supportive validation

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Experimental details: Fig. 2 Mesothelial cells phagocytose FedEVs derived from asbestos-exposed macrophages under ferroptosis. (a) Schema of culture system for the uptake by MeT-5A (recipient) cells of FedEVs from THP1 cells stably expressing GFP-CD63. (b) Fluorescent cellular images of recipient mesothelial cells phagocytosing GFP-positive FedEVs derived from THP1 macrophage cells exposed to asbestos; red, endogenous CD63; bar = 10 mum. (c) Time-course of quantitative colocalization ratio between GFP-CD63 and endogenous CD63 (mean +- SEM). (d, e) FACS analysis of MeT-5A mesothelial cells for catalytic Fe(II) (RhoNox-4, red) after exposure to GFP-CD63-labeled FedEVs derived from HT1080 by ferroptosis-stimulation with RSL3; mean +- SEM). (f, g) FACS analysis of MeT-5A mesothelial cells for catalytic Fe(II) (RhoNox-4, red) after exposure to GFP-CD63-labeled FedEVs derived from THP1 cells by ferroptosis-stimulation with [crocidolite + Fe]; mean +- SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 2

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Experimental details: Fig. 3 Non-selective proteome analysis of asbestos-induced FedEVs reveals ferritins as major components. (a) Proteomaps depicting the fold changes and associated functions for all the identified proteins within FedEVs derived from THP1 macrophage cells (NT or crocidolite + Fe exposure). (b) Relative abundance of proteins in FedEVs (crocidolite + Fe) in comparison to EVs from untreated control THP1 cells. (c) Immunoblot analysis of FedEVs from THP1 macrophage. (d) Quantification of band intensity (n = 4; mean +- SEM). (e) Immunofluorescent analysis of peritoneal wall in situ in the murine model 6 months after ip injection of 3 mg crocidolite; CD63 (FedEVs, green); FtL (ferritin light chain, red) with blue Hoechst33342 nuclear staining (bar = 50 mum). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 3

Supportive validation

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Experimental details: Fig. 6 Phagocytosis of FedEVs causes oxidative DNA base modification (8-hydroxy-2'-deoxyguanosine, 8-OHdG) and DNA double-strand breaks detected by gammaH2AX in the recipient mesothelial cells. (a) Immunofluorescent images of mesothelial cells after exposure of THP1-derived FedEVs for 24 h; green, CD63-positive FedEVs from THP1 cells; yellow, 8-OHdG; magenda, gammaH2AX with blue Hoechst33342 nuclear staining (bar = 10 mum). (b-e) FACS analysis, detecting 8-OHdG and gammaH2AX in mesothelial cells exposed to FedEVs. (f) Immunofluorescent images of mesothelial cells surrounding granuloma in vivo 6 months after ip injection of crocidolite to mice; green, gammaH2AX; yellow, CD63; magenta, FtL (bar = 20 mum at the left panels; 20 mum at the right 3 panels). (g) Quantification of the gammaH2AX in mesothelial cells. Refer to text for details (mean +- SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 6

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Experimental details: Figure 2 Western blot detection of CD9, Hsc70/Hsp70 and Calnexin in plasma pool EVs. As a positive control, lysate from the colorectal cancer cell line SW480 was used. Full-length blots are presented in Supplementary Fig. 3 (Supplementary Information).

Supportive validation

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Experimental details: Figure 2 Successful isolation and characterization of engineered EVs displaying peptides of interest. ( A ) Representative size distribution of the naive, RDG, and E626 display EVs determined by nanoparticle tracking analysis. The peak particle sizes were 106.2 nm, 98.2 nm, and 102.6 nm, respectively. ( B ) Western blot analysis of engineered EVs (RDG and E626 fusion peptide 55kDa) for the presence of EV biomarkers CD63 (30-60 kDa) and TSG101 (44 kDa), Alix (95 kDa), and peptide HA-tag. The analysis of cell lysate and engineered EVs for cellular biomarker calnexin (67KDa) is also shown. ( C ) Representative immuno-transmission electron microscopy images of naive, RDG, and E626 EVs showing gold-labeled HA on engineered EV (RDG and E626) surfaces and CD63 EV surface markers on all the EVs.

Supportive validation

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Experimental details: Figure 1 Characterization of sEVs isolated from plasma of young (3-month-old) and old mice (24-month-old). sEVs were enriched by differential ultracentrifugation. ( A ) Nanoparticle-tracking analysis (NTA) of sEV fractions from young (blue line) and old (red line) mice blood plasma. The graph shows sEV concentration and size, mean +- SEM ( n = 3). ( B ) Protein equivalents measured by microBCA. ( C ) Cryo-TEM on purified sEVs. ( D ) Western blotting for sEV- and endothelial-associated markers. ** p < 0.01 from unpaired t -test with Welch's correction.

Supportive validation

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Experimental details: 10.1371/journal.ppat.1010322.g004 Fig 4 Cytosolic entry of reovirus cores. (A) WT, KO, and KO+ HBMECs were adsorbed with Alexa 647 labeled-reovirus virions (cyan) at an MOI of 10,000 particles/cell at 37degC for 45 min and fixed with 4% PFA at 8 h post-adsorption. Cells were stained with DAPI (gray), a CD63-specific antibody to label endosomes (red), and an antiserum specific for reovirus cores (green), and imaged using confocal microscopy. Representative confocal micrographs are shown. Insets represent a merged image (far left), cores (center left), CD63 (center right), and virus (far right). (B) Colocalization of reovirus virions, cores, and endosomes was analyzed using the JaCoP plugin function from ImageJ. The results are presented as the mean colocalization (quantified by Manders coefficient) of ~ 50 cells from three independent experiments. Error bars indicate standard deviation. **, P < 0.01; ***, P < 0.001, as determined by two-tailed unpaired t-test.

Supportive validation

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Experimental details: MuVs and lmEVs present different markers and have different buoyant properties. ( A ) histograms showing the MuV and lmEV particle-size distributions (representative sample, from ALS EVs). ( B ) Representative Western blots showing the detection of CD63, CD81, CD82, AnnexinA1, ARF6, and actin, in MuVs (line1), lmEVs (line 2) and cells (line 3). Exosomal markers were enriched in MuVs and at relatively low or undetectable levels in lmEVs (EVs were extracted from the same cell culture medium for both exosomal and microparticle markers). Protein loaded on the gel is also shown, as loading control. Cellular contamination was not observed as neither of the vesicle fractions were positive for alpha-skeletal actin. ( C ) Vesicle extracts loaded on iodixanol gradients. MuVs presented classic exosomal buoyant properties while the buoyant range of lmEVs extended to a higher iodixanol density. Top panel: representative Western blot showing detection of CD63 for the MuVs at a density of 1.112 g*mL -1 ; bottom panel: representative Western blot (EVs extracted from the same cell culture medium as top panel) showing detection of Annexin A1 and ARF6 for the lmEVs at a density of 1.086-1.112 g*mL -1 and 1.185-1.230 g*mL -1 . Right panel: iodixanol/sucrose density across the 12 fractions; gradient range from 1.048 to 1.263 g*mL -1 . ( D ) Protein concentration in MuVs samples. Protein quantification was below detection sensitivity for lmEVs (ND: not detected). ( E ) Lipid quantification in MuVs

Supportive validation

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Experimental details: Characterization of human oligodendroglia-derived extracellular vesicles. A Western blotting analysis of small extracellular vesicle protein markers CD63, CD9, Flotillin-1, Annexin A2, HSP70, and the negative control marker GRP94. B Representative overview (scale bar 200 nm) and cropped (scale bar 100 nm) image showing the morphology, as revealed by transmission electron microscopy, of EVs isolated from wild-type native human oligodendroglia (OL-EVs) and stably expressing HSPB8 oligodendroglia (OL-HSPB8-EV). C - E Particle size profile distribution, particles per mL, and total EV protein measured by Nanoparticle Tracking Analysis (NTA) and BCA of OL-EVs and OL-HSPB8-EVs, pelleted from 30 mL of conditioned medium. F - H Western blotting and qPCR analysis of HSPB8 present in OL-EVs and OL-HSPB8-EVs. Statistical analysis was performed using GraphPad. Student's t-test was used to determine significance (** P < 0.01, **** P < 0.0001). Data are presented as mean +- SEM

Supportive validation

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Experimental details: 10.1371/journal.pone.0272511.g002 Fig 2 Exosome characterization. (A) Immunocytochemical detection of CD9 as an exosomal marker on GFP-packed exosome. Imaged with Fluorescence microscope at 400X total magnification. (B) Dot blot analysis of isolated exosomes for CD9 and CD63 as exosome markers. Detection of Tom40 signal for Tom40-exosome. PBS as a negative control. (C) The significant presence of Tom40 protein in isolated Tom40-exosome was shown in Western blot analysis. (D) Graphical representation of Tom40 signal normalized by CD9. Student t-test was performed *P

Supportive validation

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Experimental details: Characterization of the purified exosomes from hiPSC-EC and HCMEC conditioned media. Exosomes isolated from cell culture media of hiPSC-ECs and HCMECs. ( A ) Western blot showed the detection of exosome markers CD9, CD63, and CD81 in isolated exosomes. ( B ) Glucose-regulated protein 94 (GRP94) and Golgi marker GM130 were not found in exosomes. ( C ) Flow cytometry analysis using exosome marker CD9. ( D ) The size distribution of isolated exosomes was measured by nanoparticle tracking analysis (NTA) in hiPSC-ECs and HCMECs.

Supportive validation

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Experimental details: Characterization of EVs released by SW620, CD9-GFP + SW620, and SW480 cells. ( A - E ) EVs were recovered from the conditioned media of SW620, CD9-GFP + SW620, and SW480 cells by differential centrifugation, and the resulting 200,000x g pellets were analyzed by the ZetaView particle analyzer ( A ), immunoblotting ( B , C ), and dSTORM ( D , E ). The concentration and size of EVs derived from the indicated cells are shown ( A ). Note the presence of a common population of small particles ( 5000 EVs per experiment). Note that small and large EVs derived from SW480 cells were also quantified ( Supplementary Figure S2 ). Scale bars, 50 nm.

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Experimental details: Figure 1 CSSC cells secrete extracellular vesicles (EVs). (A): Negative staining transmission electron microscopy of a high-speed pellet from conditioned media of cultured CSSC cells shows abundant vesicles with a range of diameters from 16 to 180 nm having a median of 28 nm. (B): Size distribution of extracellular vesicles isolated from CSSC conditioned media was examined using resistive pulse sensing as described in the ""Materials and Methods"" section. (C): CD63-bead cytometry was used to identify surface antigens present on the EVs isolated from CSSC. (D): Western blotting of 10 mug protein from purified EVs from three CSSC cell lines (C1, C2, C3) and from HEK293T cells (HEK). Antibodies to calnexin (CNX) and CD63 were used on the same samples.

Supportive validation

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Experimental details: Figure 2 Extracellular vescile (EV) characterization. ( A ) Nanoparticle tracking analysis (NTA) showing a peak between 100-200 nm for the control (CT), luminal A (LA), and triple negative (TNBC) groups. ( B ) Transmission electron microscopy (TEM) image of EVs from cancer patient showing a size corresponding to NTA results. Size bar = 200 nm. ( C ) Western blotting (WB) analysis showing strong protein expression of CD9 and CD63 on EVs from control (2) and cancer (4), when compared with corresponding serum supernatant EV-depleted from control (1) and cancer (3).

Supportive validation

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Experimental details: Figure 3 Identification of S1P 2 in exosomes shed from MDA-MB-231 breast cancer cells (A) Western blot with anti-GFP or anti-mCherry antibodies showing the over-expression of GFP-hsp70 and mCherry-tgs101 in vector (V) or plasmid transfected MDA-MB-231 cells for 24 or 48 h. (B) Western blot with anti-GFP or anti-mCherry or anti-HA antibodies showing the presence of GFP-hsp70, mCherry-tsg101 or HA tagged S1P 2 (Mr = 40 kDa) in transfected MDA-MB-231 cell lysate (CL) and acid precipitates (PPT) of CM. (C) Western blot with anti-hsp70, anti-CD63 and anti-S1P 2 antibodies showing the presence of GFP-hsp70, CD63 or S1P 2 in MDA-MB-231 cell lysates (CL) and isolated exosomes (EXO) from CM. (D) Immunofluorescence image of MDA-MB-231 cells stained with anti-CD63/TRITC (red) secondary and anti-S1P 2 /FITC (green) secondary antibodies showing co-localisation (yellow) of CD63 and S1P 2 in large vesicles typical of MVBs. (E) Electron micrograph of immunogold staining with anti-CD63 [attached to secondary goat anti-mouse IgG-immune-gold particles (15 nm)] and anti-S1P 2 antibodies [attached to secondary goat anti-rabbit IgG-immune-gold particles (10 nm)] in exosomes isolated from CM of MDA-MB-231 cells. Control represents exosomes that have not been incubated with primary antibody. Results are representative of 3 independent experiments.

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Experimental details: Fig. 2 EV biogenesis is impaired in C9-ALS iAstrocytes. Representative size distribution graph (a) of ADEVs and TEM (b, c-i, c-ii) show that most ADEVs range between 50 and 120 nm. ADEVs are positive for CD63 (arrow heads) with immunogold labeling (c-iii). ADEV direct quantification using the ZetaView Nanoparticle Tracking System (d) shows a significant decrease in the number of particles secreted by C9 iAstrocytes (One-way ANOVA; p < .01). This suggests impairment in vesicle formation, as supported by TaqMan qRT-PCR data showing downregulation in a number of transcripts involved in EV biogenesis in C9 iAstrocytes (e). t -test (**p < .01; *** p < .001), 3xControls versus 3xC9-ALS, N = 3 per condition, error bar = SD. Fig. 2