CiteAb

Fig.4.A showing Western Blotting from the publication: Endoplasmic reticulum stress alters myelin associated protein expression and extracellular vesicle composition in human oligodendrocytes.

Fig.3.D showing Western Blotting from the publication: Endoplasmic reticulum stress alters myelin associated protein expression and extracellular vesicle composition in human oligodendrocytes.

Fig.2.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Inline Raman Spectroscopy Provides Versatile Molecular Monitoring for Platelet Extracellular Vesicle Purification with Anion-Exchange Chromatography.

Fig.3.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Inline Raman Spectroscopy Provides Versatile Molecular Monitoring for Platelet Extracellular Vesicle Purification with Anion-Exchange Chromatography.

Fig.1.E showing Western Blotting from the publication: Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Fig.2.E showing Western Blotting from the publication: Exosomal release of the virus-encoded chemokine receptor US28 contributes to chemokine scavenging.

Fig.2.A showing Immunocytochemistry-immunofluorescence from the publication: Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages.

Fig.6.B showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Fig.6.F showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Fig.6.D showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Fig.6.A showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Fig.1.C showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Fig.1.M showing Western Blotting from the publication: Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses.

Fig.1.A showing Western Blotting from the publication: Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

Fig.2.C showing Western Blotting from the publication: Transforming growth factor-β-induced secretion of extracellular vesicles from oral cancer cells evokes endothelial barrier instability via endothelial-mesenchymal transition.

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.1.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.8.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.3.B showing Immunocytochemistry-immunofluorescence from the publication: PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers.

Fig.1.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers.

Fig.6.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Novel Platform for Regulation of Extracellular Vesicles and Metabolites Secretion from Cells Using a Multi-Linkable Horizontal Co-Culture Plate.

Fig.5.B showing Western Blotting from the publication: The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

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Extracellular vesicles from different regions of the female reproductive tract promote spermatozoa motility and support capacitation.

In Scientific Reports on 17 July 2025 by Fasoli, C., Giacomini, E., et al.

Extracellular vesicles (EVs) have been proven to have a crucial role in intercellular communication and have attracted significant attention in the physiology of reproduction because of their multiple functions in physiological processes essential for reproduction including gametogenesis, fertilization and embryo-endometrial cross-talk. Although EVs from the male reproductive tract have been extensively studied for their role in sperm maturation, research on female reproductive tract-derived EVs in humans is still emerging and supported by only a few studies to date. In vitro study was performed using spermatozoa from normozoospermic men and EVs isolated from follicular fluid (FF-EVs), cervicovaginal fluid collected 2 and 7 days after the LH surge (CVF-EVs LH + 2 and LH + 7, respectively) and spent medium of decidualized (dESCs-EVs) and non-decidualized (eESCs-EVs) endometrial stromal cells from healthy women of reproductive age. The principal outcome measures comprise the percentage of viable, progressively motile, and capacitated spermatozoa after treatment with FF-EVs, CVF-EVs LH + 2 and LH + 7, dESCs-EVs, and eESCs-EVs. Spermatozoa are able to capture EVs derived from all the considered tracts of the female reproductive system, with slightly varying efficiencies, albeit comparable in most cases. Incubating sperm cells with any of these EVs does not have any detrimental effect on sperm vitality, increases the percentage of spermatozoa displaying progressive motility and the percentage of acrosome-reacted spermatozoa. EVs produced and released in various regions of the female reproductive system likely contribute to spermatozoa maturation during their transit, promoting both capacitation and motility.
© 2025. The Author(s).

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Calpains orchestrate secretion of annexin-containing microvesicles during membrane repair.

In The Journal of Cell Biology on 7 July 2025 by Williams, J. K., Ngo, J. M., et al.

Microvesicles (MVs) are membrane-enclosed, plasma membrane-derived particles released by cells from all branches of life. MVs have utility as disease biomarkers and may participate in intercellular communication; however, physiological processes that induce their secretion are not known. Here, we isolate and characterize annexin-containing MVs and show that these vesicles are secreted in response to the calcium influx caused by membrane damage. The annexins in these vesicles are cleaved by calpains. After plasma membrane injury, cytoplasmic calcium-bound annexins are rapidly recruited to the plasma membrane and form a scab-like structure at the lesion. In a second phase, recruited annexins are cleaved by calpains-1/2, disabling membrane scabbing. Cleavage promotes annexin secretion within MVs. Our data support a new model of plasma membrane repair, where calpains relax annexin-membrane aggregates in the lesion repair scab, allowing secretion of damaged membrane and annexins as MVs. We anticipate that cells experiencing plasma membrane damage, including muscle and metastatic cancer cells, secrete these MVs at elevated levels.
© 2025 Williams et al.

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ADAM Sheddase Activity Promotes the Detachment of Small Extracellular Vesicles From the Plasma Membrane.

In Journal of Extracellular Vesicles on 1 July 2025 by Bizingre, C., Arellano-Anaya, Z., et al.

Small extracellular vesicles (SEVs) are involved in diverse functions in normal and pathological situations, including intercellular communication, immunity, metastasis and neurodegeneration. Cell release of SEVs is assumed to occur passively right after multivesicular bodies of the endocytic pathway fuse with the plasma membrane. We show here that the completion of SEV release depends on membrane-bound ADAM10 and ADAM17 sheddases that promote the detachment of SEVs from the cell surface by catalysing the cleavage of adhesion proteins of the SEV membrane. The intensity of ADAM10/17-mediated release of SEVs depends on a balanced control of 3-phosphoinositide-dependent kinase 1 (PDK1) and ERK1/2 signalling pathways converging on 90-kDa ribosomal S6 kinase-2 (RSK2), which, in turn, fine-tunes ADAM17 bioavailability and ADAM10/17 enzymatic activities at the plasma membrane, according to a mechanism that relies, at least in part, on variation of the rhomboid-like pseudoprotease iRhom2 cell surface level. By identifying a new proteolytic step involved in the basal release of SEVs, our work may help understand how the deregulation of ADAM10/17-mediated discharge of SEVs contributes to several pathological states.
© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.

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A Simple and Scalable Assay for Multiplexed Flow Cytometric Profiling of Surface Markers on Small Extracellular Vesicles.

In Cells on 28 June 2025 by Polignano, D., Barreca, V., et al.

Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, play crucial roles in intercellular communication and disease pathology. Their heterogeneous nature, shaped by cellular origin and activation state, requires precise and multiplexed profiling of surface markers for effective characterization. Despite recent advances, current analytical methods remain complex, costly, or inaccessible for routine laboratory use. Here, we present a simple and cost-effective flow cytometry-based assay for the multiplexed analysis of tetraspanin markers (CD63, CD81, CD9) on fluorescently labeled sEVs. Our method combines metabolic labeling with paraformaldehyde fixation and low-speed centrifugation using a benchtop centrifuge, enabling efficient removal of unbound antibodies and minimizing nonspecific signals while preserving vesicle integrity. Using either metabolically labeled exosomes or bulk sEVs stained with carboxyfluorescein succinimidyl ester (CFSE), we demonstrate robust recovery and accurate, semi-quantitative profiling of tetraspanin expression. The assay reveals substantial variability in tetraspanin distribution across different cell lines and does not require ultracentrifugation or immunocapture. Notably, this versatile and reproducible method supports high sEV recovery and is adaptable to additional protein markers. Its compatibility with standard laboratory equipment makes it a practical and scalable alternative to more complex techniques, expanding access to multiplex sEV analysis for both research and clinical applications.

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Characterization of a novel FLI1 mutation in a family with thrombocytopenia and other congenital malformations.

In Haematologica on 5 June 2025 by Ammeti, D., Barozzi, S., et al.

Not available.

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Inline Raman Spectroscopy Provides Versatile Molecular Monitoring for Platelet Extracellular Vesicle Purification with Anion-Exchange Chromatography.

In Int J Mol Sci on 25 July 2024 by Saari, H., Marttila, H., et al.

Fig.2.D

Fig.2.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Inline Raman Spectroscopy Provides Versatile Molecular Monitoring for Platelet Extracellular Vesicle Purification with Anion-Exchange Chromatography.

Monolithic anion-exchange chromatography separates EVs from most of the contaminants in platelet supernatant. (A) Representative chromatogram of each sample type (standard, high load, and added BSA), monitored with a UV detector at 280 nm. The flowthrough (FT) included sample injection into the c... more

Monolithic anion-exchange chromatography separates EVs from most of the contaminants in platelet supernatant. (A) Representative chromatogram of each sample type (standard, high load, and added BSA), monitored with a UV detector at 280 nm. The flowthrough (FT) included sample injection into the column and washing of unbound material. FT was collected into one fraction for downstream analysis. Bound material was eluted with a high-salt buffer, indicated by an increase in conductivity, and 1 mL fractions were collected. Fraction 1 was column void volume and not collected. (B,C) Assessment of EV marker distribution (CD9 in B and CD63 in (C) in the fractions using Western blotting. Equal volume loading was used to normalize the sample volume in the assay, representing the total yields in each fraction. (D) A representative blot, including ApoB staining also, where only the ApoB-100 isoform is visible. (E) Nanoparticle distribution in the fractions, as assessed using Nanoparticle Tracking Analysis. The results in B, C, and E were normalized against the total sum of marker intensity or particle yield in the fractions of each individual run. The results in (C–E) are shown with the mean normalized intensity ± standard error. Statistical significance was tested using a two-way ANOVA with Fisher’s LSD post hoc test and denoted by an asterisk: * p < 0.05 (n = 6 for each sample type). Only significant differences due to the sample type are denoted. less

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Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

In Commun Biol on 6 May 2024 by Fujita, Y., Kadota, T., et al.

Fig.1.E

Fig.1.E showing Western Blotting from the publication: Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Characterization and functions of HBEC-EVs.(A) Cryotransmission electron microscopy of purified HBEC-EVs using ultracentrifugation. (B) Nanoparticle tracking analysis displaying the particle size of purified HBEC-EVs. The vertical axis in the graph represents the number of EV particles (x 108)/mL... more

Characterization and functions of HBEC-EVs.(A) Cryotransmission electron microscopy of purified HBEC-EVs using ultracentrifugation. (B) Nanoparticle tracking analysis displaying the particle size of purified HBEC-EVs. The vertical axis in the graph represents the number of EV particles (x 108)/mL, and the horizontal axis indicates the particle size (nm) of EVs. (C) Analysis of HBEC-EVs using a bioanalyzer. Gels and electropherogram are presented. The left gel lane is the ladder standard, and the right lane contains the total RNA from HBEC-EVs. The y-axis of the electropherogram displays signal intensities in arbitrary fluorescence units (FU), and the x-axis indicates the size of the RNA in nucleotides (nt). (D) Utilization of a multiplex bead-based flow cytometry assay to detect HBEC-EV surface signatures. Thirty-seven multiplexed populations of dye-labeled antibody-coated capture beads were incubated with three different HBEC-EV samples. The mean fluorescence intensity (MFI) of each marker was analyzed. ND: not detected. (E) Western blots of HBECs and HBEC-EVs for CD9, CD63, CD81, MHC-2, and β-actin. (F-G) Measurement of cytokine secretion in HBECs at 8 h after poly (I:C) (250 ng/ml) and HBEC-EV administration (10 μg/ml) (F) and in THP-1 cells at 24 h after LPS (1 μg/ml) and HBEC-EV administration (10 μg/ml) (G). THP-1 cells were treated with 100 nM PMA for 24 h before the experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. NS; not significant. less

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Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.

Fig.6.B

Fig.6.B showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells t... more

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct. Proteins were detected from gels with sample loading normalised to total cell lysate protein levels and the fold change in CHMP5 knockdown cells determined. Note the more variable effects of knockdown on some of the sEV markers, but not Rab11a, in the bar chart. This is presumably the result of different levels of transient CHMP5 knockdown. Data derived from three independent experiments. (B) Nanosight tracking analysis of sEVs for diluted samples (normalised to cell lysate protein levels) shows no change in particle size and number. (C) Western blot analysis of putative exosome proteins from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct shows selective reduction in CHMP5 protein levels. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1, using phospho‐specific antibodies and a pan 4E‐BP1 antibody. Bar chart shows protein levels normalised to tubulin. Data derived from three independent experiments. (D) Western analysis of sEV preparations isolated by SEC from HCT116 colorectal cancer cells, which contain a stable IPTG‐inducible CHMP5 shRNA knockdown construct (clone #28; shCHMP5 #28), cultured in glutamine‐depleted (0.15 mM) conditions for 24 h. sEVs were collected from cells cultured in the absence (‐) or presence (+) of IPTG, both for 96 h previously and during the collection period. Putative exosome proteins were detected from gels with sample loading normalised to total cell lysate protein levels. Bar charts represent changes in levels of these putative exosome proteins relative to levels in the non‐IPTG‐induced sample. Data derived from five independent experiments. (E) Nanosight Tracking Analysis of sEVs for the shCHMP5 #28 samples produced in (D) show no change in particle size and number. (F) Western blot analysis of putative exosome proteins in EV‐secreting cells carrying a stable IPTG‐inducible CHMP5 (shCHMP5 #28) shRNA construct, under glutamine‐depleted (0.15 mM) conditions, in the presence or absence of IPTG, reveals selective reduction in CHMP5 protein levels. Bar charts derived from three independent experiments. All data for bar charts were analysed by the Kruskal‐Wallis test: *p < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD less

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Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.

Fig.6.F

Fig.6.F showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells t... more

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct. Proteins were detected from gels with sample loading normalised to total cell lysate protein levels and the fold change in CHMP5 knockdown cells determined. Note the more variable effects of knockdown on some of the sEV markers, but not Rab11a, in the bar chart. This is presumably the result of different levels of transient CHMP5 knockdown. Data derived from three independent experiments. (B) Nanosight tracking analysis of sEVs for diluted samples (normalised to cell lysate protein levels) shows no change in particle size and number. (C) Western blot analysis of putative exosome proteins from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct shows selective reduction in CHMP5 protein levels. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1, using phospho‐specific antibodies and a pan 4E‐BP1 antibody. Bar chart shows protein levels normalised to tubulin. Data derived from three independent experiments. (D) Western analysis of sEV preparations isolated by SEC from HCT116 colorectal cancer cells, which contain a stable IPTG‐inducible CHMP5 shRNA knockdown construct (clone #28; shCHMP5 #28), cultured in glutamine‐depleted (0.15 mM) conditions for 24 h. sEVs were collected from cells cultured in the absence (‐) or presence (+) of IPTG, both for 96 h previously and during the collection period. Putative exosome proteins were detected from gels with sample loading normalised to total cell lysate protein levels. Bar charts represent changes in levels of these putative exosome proteins relative to levels in the non‐IPTG‐induced sample. Data derived from five independent experiments. (E) Nanosight Tracking Analysis of sEVs for the shCHMP5 #28 samples produced in (D) show no change in particle size and number. (F) Western blot analysis of putative exosome proteins in EV‐secreting cells carrying a stable IPTG‐inducible CHMP5 (shCHMP5 #28) shRNA construct, under glutamine‐depleted (0.15 mM) conditions, in the presence or absence of IPTG, reveals selective reduction in CHMP5 protein levels. Bar charts derived from three independent experiments. All data for bar charts were analysed by the Kruskal‐Wallis test: *p < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD less

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Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.

Fig.6.D

Fig.6.D showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells t... more

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct. Proteins were detected from gels with sample loading normalised to total cell lysate protein levels and the fold change in CHMP5 knockdown cells determined. Note the more variable effects of knockdown on some of the sEV markers, but not Rab11a, in the bar chart. This is presumably the result of different levels of transient CHMP5 knockdown. Data derived from three independent experiments. (B) Nanosight tracking analysis of sEVs for diluted samples (normalised to cell lysate protein levels) shows no change in particle size and number. (C) Western blot analysis of putative exosome proteins from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct shows selective reduction in CHMP5 protein levels. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1, using phospho‐specific antibodies and a pan 4E‐BP1 antibody. Bar chart shows protein levels normalised to tubulin. Data derived from three independent experiments. (D) Western analysis of sEV preparations isolated by SEC from HCT116 colorectal cancer cells, which contain a stable IPTG‐inducible CHMP5 shRNA knockdown construct (clone #28; shCHMP5 #28), cultured in glutamine‐depleted (0.15 mM) conditions for 24 h. sEVs were collected from cells cultured in the absence (‐) or presence (+) of IPTG, both for 96 h previously and during the collection period. Putative exosome proteins were detected from gels with sample loading normalised to total cell lysate protein levels. Bar charts represent changes in levels of these putative exosome proteins relative to levels in the non‐IPTG‐induced sample. Data derived from five independent experiments. (E) Nanosight Tracking Analysis of sEVs for the shCHMP5 #28 samples produced in (D) show no change in particle size and number. (F) Western blot analysis of putative exosome proteins in EV‐secreting cells carrying a stable IPTG‐inducible CHMP5 (shCHMP5 #28) shRNA construct, under glutamine‐depleted (0.15 mM) conditions, in the presence or absence of IPTG, reveals selective reduction in CHMP5 protein levels. Bar charts derived from three independent experiments. All data for bar charts were analysed by the Kruskal‐Wallis test: *p < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD less

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Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.

Fig.6.A

Fig.6.A showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells t... more

Inducible knockdown of CHMP5 selectively reduces Rab11a‐exosome production from glutamine‐depleted HCT116 cells. (a) Western blot analysis of putative exosome proteins in sEV preparations isolated by size‐exclusion chromatography (SEC) from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct. Proteins were detected from gels with sample loading normalised to total cell lysate protein levels and the fold change in CHMP5 knockdown cells determined. Note the more variable effects of knockdown on some of the sEV markers, but not Rab11a, in the bar chart. This is presumably the result of different levels of transient CHMP5 knockdown. Data derived from three independent experiments. (B) Nanosight tracking analysis of sEVs for diluted samples (normalised to cell lysate protein levels) shows no change in particle size and number. (C) Western blot analysis of putative exosome proteins from glutamine‐depleted (0.15 mM) EV‐secreting HCT116 cells transduced with a CHMP5 (shCHMP5) compared to a non‐targeting (shNT) shRNA construct shows selective reduction in CHMP5 protein levels. The activity of mTORC1 was assessed via phosphorylation of S6 and 4E‐BP1, using phospho‐specific antibodies and a pan 4E‐BP1 antibody. Bar chart shows protein levels normalised to tubulin. Data derived from three independent experiments. (D) Western analysis of sEV preparations isolated by SEC from HCT116 colorectal cancer cells, which contain a stable IPTG‐inducible CHMP5 shRNA knockdown construct (clone #28; shCHMP5 #28), cultured in glutamine‐depleted (0.15 mM) conditions for 24 h. sEVs were collected from cells cultured in the absence (‐) or presence (+) of IPTG, both for 96 h previously and during the collection period. Putative exosome proteins were detected from gels with sample loading normalised to total cell lysate protein levels. Bar charts represent changes in levels of these putative exosome proteins relative to levels in the non‐IPTG‐induced sample. Data derived from five independent experiments. (E) Nanosight Tracking Analysis of sEVs for the shCHMP5 #28 samples produced in (D) show no change in particle size and number. (F) Western blot analysis of putative exosome proteins in EV‐secreting cells carrying a stable IPTG‐inducible CHMP5 (shCHMP5 #28) shRNA construct, under glutamine‐depleted (0.15 mM) conditions, in the presence or absence of IPTG, reveals selective reduction in CHMP5 protein levels. Bar charts derived from three independent experiments. All data for bar charts were analysed by the Kruskal‐Wallis test: *p < 0.05; n.s. = not significant. Bars and error bars denote mean ± SD less

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Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.

Fig.1.C

Fig.1.C showing Western Blotting from the publication: Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

sEV preparations from glutamine‐depleted HCT116 colorectal cancer cells contain elevated levels of accessory ESCRT‐III proteins. Five paired protein samples made from sEVs isolated by differential ultracentrifugation from glutamine‐depleted and glutamine‐replete HCT116 cells were subjected to TMT... more

sEV preparations from glutamine‐depleted HCT116 colorectal cancer cells contain elevated levels of accessory ESCRT‐III proteins. Five paired protein samples made from sEVs isolated by differential ultracentrifugation from glutamine‐depleted and glutamine‐replete HCT116 cells were subjected to TMT‐labelled comparative proteomics analysis. (a) Volcano plot showing mean log2 fold change in specific protein levels in sEV preparations from glutamine‐depleted conditions versus glutamine‐replete conditions plotted against ‐log10p value for null hypothesis that levels are unchanged. Coloured data points represent proteins that are significantly altered in levels between the two conditions, with colours representing number of peptides detected. (b) Heat map showing change in levels of core and accessory ESCRT proteins in five paired sEV samples (labelled 1 to 5) from glutamine‐depleted versus glutamine‐replete HCT116 cells. Red shows an increase in protein levels and blue indicates a reduction following glutamine depletion. (c) Western blot of sEV preparations confirms that accessory ESCRT‐III proteins CHMP1A, CHMP1B and IST1 are all increased in sEVs produced by glutamine‐depleted (0.15 mM) HCT116 cells. Bar chart shows protein levels and sEV number normalised to cell lysate. Data derived from five independent experiments and analysed by the Kruskal‐Wallis test. Significantly changed levels are denoted by a blue (decreased) and red (increased) asterisks. *p < 0.05; n.s. = not significant. (d) Nanosight Tracking Analysis of sEV size and number for samples produced as in (c) less

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Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses.

In Blood Cancer Discov on 6 January 2023 by Gargiulo, E., Viry, E., et al.

Fig.1.M

Fig.1.M showing Western Blotting from the publication: Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses.

sEV are enriched in the human and murine leukemic microenvironments. A, Relative mRNA expression of selected genes involved in sEV biogenesis and secretion in B cells from PB of healthy donors (HC, n = 9) and CLL patients (CLL, n = 15; from GSE67640). B, Score based on sEV-related mRNA levels fro... more

sEV are enriched in the human and murine leukemic microenvironments. A, Relative mRNA expression of selected genes involved in sEV biogenesis and secretion in B cells from PB of healthy donors (HC, n = 9) and CLL patients (CLL, n = 15; from GSE67640). B, Score based on sEV-related mRNA levels from A. C, mRNA levels of selected genes extracted from A. D, mRNA expression of selected genes according to IGHV mutational status. E, Score combining the expression of Rab10, Rab35, and Rab40C according to IGHV mutational status. F, Relative mRNA expression of selected genes involved in sEV biogenesis and secretion in B cells from C57BL/6 (WT) and Eμ-TCL1 mice (TCL1; from GSE175564). G, Score based on sEV-related RNA levels from F. H,Rab3b mRNA level extracted from F. I, Detailed protocol to isolate and purify sEVs from the murine spleen. J, Amount of proteins (in mg) recovered from LME- (n = 18) or HCME-sEVs (n = 10), normalized per gram of spleen. K, Representative TRPS analysis of ME-sEVs for size and concentration. L, Electron microscopy images of ME-sEVs. M, Western blot analysis of ME-sEVs. N, HSNE clustering analysis of MB488+ LME-sEVs based on CD63, CD81, and CD9 expression measured by bead-free FC (left) and relative percentages of combined expression (right). *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (unpaired Student t test). Data are mean. less

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Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

In Sci Rep on 22 December 2022 by Petit, I., Levy, A., et al.

Fig.1.A

Fig.1.A showing Western Blotting from the publication: Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

FGF2 is found at the outer surface of EVs secreted by dermal fibroblasts cultured in FGF2-containing medium. (A) FGF2, syntenin-1 and CD63 expression in DF cells and EV lysate (20 μg) was examined by western blot. Three isoforms of endogenous FGF2 were present in DF cells while FGF2-EVs contained... more

FGF2 is found at the outer surface of EVs secreted by dermal fibroblasts cultured in FGF2-containing medium. (A) FGF2, syntenin-1 and CD63 expression in DF cells and EV lysate (20 μg) was examined by western blot. Three isoforms of endogenous FGF2 were present in DF cells while FGF2-EVs contained the low molecular weight FGF2 isoform corresponding to recombinant FGF2. Original blots are shown in Suppl. Information. (B) Cells were cultured in the presence of His-tagged FGF2; western blot with 15 μg lysate and using anti-His antibody revealed the presence of His-FGF2 in secreted EVs. A shift in FGF2 size was also observed. Syntenin-1 was used as EV marker. Original blots are shown in Suppl. Information. (C) FGF2-EVs were loaded on a SEC column and collected fractions 8 and 9 (3E + 9p) were analyzed by western blot using antibodies for FGF2 and the EV markers CD63, CD81 and syntenin-1. Original blots are shown in Suppl. Information. For quantification, fractions were also analyzed by NTA. (D) FGF2 detection in FGF2-EVs by ELISA assay. Intact CTL- and FGF2-EVs (8E + 8p) were directly placed on FGF2 ELISA wells for external surface detection. FGF2-EVs were also treated with 0.1% triton and analyzed to detect internal FGF2. No FGF2 signal was detected in CTL-EVs. (E) FGF2 expression on FGF2-EVs was detected by flow cytometry. EVs were coupled to latex beads and labeled with FGF2 antibody before analysis by cytometry. Beads without EVs (beads) and beads labelled with goat IgG antibody (IgG) serve as negative controls. A representative plot is shown. less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.2.B

Fig.2.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

KIT‐containing EVs can be isolated by immunocapture. (A) Illustration of the predicted topology of the receptor KIT in an EV. KIT is embedded in the EV membrane by its transmembrane domain (TMD) with the N‐terminal (N‐t) ligand‐binding domain exposed to the extravesicular environment, while the C... more

KIT‐containing EVs can be isolated by immunocapture. (A) Illustration of the predicted topology of the receptor KIT in an EV. KIT is embedded in the EV membrane by its transmembrane domain (TMD) with the N‐terminal (N‐t) ligand‐binding domain exposed to the extravesicular environment, while the C‐terminal (C‐t) protein domain is directed into the EV lumen. (B) Representative immunoblots showing the canonical EV marker content of KIT‐EVs captured from HMC‐1.1‐derived EVs that were isolated by size exclusion chromatography (SEC). Three different antibodies directed against N‐t, C‐t (left panel) or full‐length (fl) KIT protein (right panel) were used for immunocapturing of KIT‐containing EVs. Each pulldown was performed from vesicles secreted by 20 × 106 cells and collected in SEC fractions seven to nine. Unbound EVs in the flow‐through (FT) were pelleted by ultracentrifugation (UC) to demonstrate the capture efficiency. Beads conjugated to isotype control antibodies served as negative controls. Cell lysates (30 μg) were included. g, goat; rb, rabbit; m, mouse. Dashed lines imply lanes from the same blot that were not loaded contiguously. (C) Schematic of the capture of KIT‐EVs by electron microscopy (EM) KIT immunocapture grids. EM grids were coated with a primary KIT antibody (targeting the N‐terminus of KIT) followed by BSA‐blocking and then incubation with PEG‐precipitated EV preparations. Captured KIT‐EVs were visualized by negative stain EM. (D) Representative images of HMC‐1.1‐released vesicles immunocaptured on KIT affinity grids and visualized by negative stain EM. A field of view containing several KIT‐EVs (blue arrows) is shown above, and a montage of KIT‐EVs below. Scale bars are indicated. (E) Size distribution of immunocaptured EVs on KIT affinity grids presented as percentage. A total of 210 EVs (single and grouped vesicles) was counted less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.1.F

Fig.1.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐... more

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐PAGE, blotted and probed with the indicated antibodies. (C) HMC‐1.1‐derived EVs were separated from soluble protein by size exclusion chromatography (SEC; qEVoriginal70) and eluted as pooled fractions F7‐F11, followed by filter concentration. Cell (30 μg) and EV lysates (15 μg) were analyzed by immunoblotting with the indicated antibodies. The dashed line indicates the samples were run on the same blot, but not in contiguous lanes. (D) A representative graph showing the concentration and size measurement of SEC‐isolated EVs (C) by nanoparticle tracking. The average profile of five workflow repetitions is displayed. (E) Workflow of the immunocapture of EV subpopulations. EVs are bulk‐isolated from pre‐cleared cell culture media by PEG‐precipitation and incubated with antibody‐conjugated beads allowing for immunocapture of specific EV subpopulations. (F) Representative Western blot of CD9‐, CD81‐ and CD63‐immunocaptured EVs. Each bead capture was performed from an equal number of HMC‐1.1‐derived EVs (1.9 × 1010 EVs). Unconjugated beads served as a negative control. The presence of canonical EV protein markers as well as KIT in the EV subpopulations were determined by immunoblotting. *, signal from previous probing with a flotillin‐1 antibody less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.1.A

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐... more

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐PAGE, blotted and probed with the indicated antibodies. (C) HMC‐1.1‐derived EVs were separated from soluble protein by size exclusion chromatography (SEC; qEVoriginal70) and eluted as pooled fractions F7‐F11, followed by filter concentration. Cell (30 μg) and EV lysates (15 μg) were analyzed by immunoblotting with the indicated antibodies. The dashed line indicates the samples were run on the same blot, but not in contiguous lanes. (D) A representative graph showing the concentration and size measurement of SEC‐isolated EVs (C) by nanoparticle tracking. The average profile of five workflow repetitions is displayed. (E) Workflow of the immunocapture of EV subpopulations. EVs are bulk‐isolated from pre‐cleared cell culture media by PEG‐precipitation and incubated with antibody‐conjugated beads allowing for immunocapture of specific EV subpopulations. (F) Representative Western blot of CD9‐, CD81‐ and CD63‐immunocaptured EVs. Each bead capture was performed from an equal number of HMC‐1.1‐derived EVs (1.9 × 1010 EVs). Unconjugated beads served as a negative control. The presence of canonical EV protein markers as well as KIT in the EV subpopulations were determined by immunoblotting. *, signal from previous probing with a flotillin‐1 antibody less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.5.B

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Mast cells produce microvesicle (P15)‐ and exosome (P120)‐like KIT‐EVs. (A) Schematic of KIT immunoprecipitation from P15 and P120 EV populations. (B) KIT‐EVs were purified from P15 or P120 EVs (8.9 × 109 EVs) by immunoprecipitation with an antibody against N‐terminal KIT. EVs in the flow‐through... more

Mast cells produce microvesicle (P15)‐ and exosome (P120)‐like KIT‐EVs. (A) Schematic of KIT immunoprecipitation from P15 and P120 EV populations. (B) KIT‐EVs were purified from P15 or P120 EVs (8.9 × 109 EVs) by immunoprecipitation with an antibody against N‐terminal KIT. EVs in the flow‐through were captured with CD9‐conjugated beads. Unconjugated beads and a mix of P15 and P120 vesicles were used as a negative control. Cell lysates (30 μg) and EV eluates were analyzed by Western blotting with the indicated antibodies. Representative blots are shown. *, indicates bands arising from protein G. (C) Relative signal intensities of protein markers shown in (B) were quantified and normalized to signals in P15 KIT‐EVs. Data are shown as mean ± S.D. from three independent experiments. Proteins enriched in P120 and P15 KIT‐EVs are highlighted in orange and blue, respectively. Ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired Student t‐test). (D) The concentration of P15 and P120 EVs before and after KIT immunoprecipitation shown in (B) was measured by nanoparticle tracking. The representative profile shows the distribution of particle numbers for a range of particle sizes averaged from five workflow repetitions. The sizes (nm) of the most abundant particles in P15 and P120 are indicated. The percentage of KIT‐EVs was estimated by determining the particle count before and after KIT‐EV capturing. FT, flow through. (E) After SILAC labelling of HMC‐1.1 cells with heavy and light amino acids, EVs were isolated from the cell culture supernatant (SN) by ultracentrifugation as P15 and P120 EV pellets. EV suspensions were further cleaned by size exclusion chromatography (SEC) before performing KIT immunocapturing on each EV pool. Heavy‐labeled KIT P15 EVs were mixed with light‐labelled KIT P120 EVs (and vice versa) and processed for quantitative mass spectrometry as inverse duplicates. (F) The enrichment of exemplary proteins (presented as log2 fold change H/L) determined by SILAC mass spectrometry in P15 KIT‐EVs compared to P120 KIT‐EVs is shown. Data are presented as the mean from two mass spectrometry pairs ± S.D. The red line indicates the 2‐fold enrichment threshold. Proteins highly enriched in P15 KIT‐EVs are indicated in blue. The ‐Log T‐test p‐value is shown above each bar. ‐Log T‐test p‐values > 1.3 indicate significance. (G) Illustration of P15 and P120 KIT‐EVs summarizing the association of EV markers with respective vesicle types. Protein markers highly associated with either P15 (microvesicle‐like) or P120 (exosome‐like) EVs are highlighted in red less

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The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

In Cancers (Basel) on 26 October 2021 by Chen, Y. Y., Dagg, R., et al.

Fig.5.B

Fig.5.B showing Western Blotting from the publication: The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Immuno-purification of C-Circles with exosomes: (a) schema of the CD81 immunopurification method, fractions with names in bold font are analyzed by the C-Circle assay (CCA). (b) Representative CCA dot blot of the immunopurification fractions, with the sample-specific background (Background) check... more

Immuno-purification of C-Circles with exosomes: (a) schema of the CD81 immunopurification method, fractions with names in bold font are analyzed by the C-Circle assay (CCA). (b) Representative CCA dot blot of the immunopurification fractions, with the sample-specific background (Background) checked by omitting the Phi29 DNA polymerase from the CCA. (c) Quantitation (n = 3) of the CCA presented as the percentage of the total CCA signal (CD81-PD and CD81-FT combined). (d) Representative Western blot of exosome-specific proteins CD9, CD63, CD81, and Syntenin in the CD81-PD and CD81-FT fractions. The positions of the protein size standards are shown. (e) Quantitation (n = 3) of the Western blots presented as the percentage of the total combined signal. CD81-PD or CD81-FT are treated with Dnase I either before (pre-extraction) or after (post-extraction) the extraction of DNA for the CCA. A representative dot blot is shown (f), with whole cell DNA (Cell-DNA) and sample-specific background (Background) controls. The quantitation of the replicates (n = 2) is plotted in (g), which shows that before DNA extraction, 25% (SEM = 9%) and 60% (SEM = 8%) of the CCA signal in the CD81-PD and CD81-FT, respectively, are accessible to DNAse I. All error bars indicate the SEM. Overexposed CCA dot blots are presented in Figure S3 for (b,f) to allow for better visualization of the Background signal. ** p < 0.002 and *** p < 0.0003 by the Friedman test. less

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