Introduction: Exosomes play significant roles in transferring cargo materials like proteins, RNAs (including miRNAs), and DNA. However, the role of serum exosome shuttled RNAs and miRNAs in head and neck cancer (HNC) remains unclear. This study assessed the diagnostic and prognostic significance of exosomal miR-17, miR-20a, and TGFBR2 in HNC patients. Methods: Exosomes were isolated, from 400 confirmed HNC patients and 400 healthy controls, and characterized by NTA, TEM, Immunolabelling, and ELISA. Quantitative PCR was used to check the expressions of exosomal molecules. Oxidative stress was also measured through ELISA in cancer patients and healthy controls. Results: Data analysis revealed significant dysregulation in the expressional levels of miR-17 (p < .0001), miR-20a (p = .0003), and TGFBR2 (p = .0005), which were found associated with aggressiveness and poor survival of HNC patients. Spearman correlation revealed a positive statistically significant association between miR-20a versus miR-17 (r = 0.534; p < .01), while a negative correlation was found between TGFBR2 versus miR-17 (r = -0.240; p = .015). Significantly decreased levels of peroxidase (POD) (p < .0001) and an increased level of 8-Oxoguanine (p < .0001) were observed. Conclusion: The results showed that these exosomal miRNAs and target gene may serve as potential and noninvasive diagnostic and prognostic markers for head and neck cancer patients.
Product Citations: 325
Deregulation of Exosomal miR-17, miR-20a and TGFBR2 in Head and Neck Cancer Patients.
In Technology in Cancer Research Treatment on 24 February 2025 by Rizwan, M., Mahjabeen, I., et al.
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Cancer Research
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In Cellular and Molecular Life Sciences : CMLS on 13 February 2025 by Picard, F., Nonaka, T., et al.
Proteinopathies, such as amyotrophic lateral sclerosis (ALS), are marked by the accumulation of misfolded proteins that disrupt cellular processes. Eukaryotic cells have developed protein quality control systems to eliminate these aberrant proteins, but these systems often fail to differentiate between normal and misfolded proteins. In ALS, pathological inclusions primarily composed of misfolded TDP-43 are a hallmark of the disease. Recently, a novel unconventional secretion process called misfolding-associated protein secretion (MAPS) has been discovered to selectively export misfolded proteins. USP19, an Endoplasmic Reticulum-associated ubiquitin peptidase, plays a crucial role in this process. In this study, we investigated the impact of ER-anchored USP19 on the secretion of misfolded TDP-43. Here we found that USP19 overexpression significantly promotes the secretion of soluble and aggregated misfolded TDP-43, requiring both ER anchoring and ubiquitin peptidase activity. Characterization of the cellular and molecular mechanisms involved in this process highlighted the importance of early autophagosomal and late endosomal/amphisomal compartments, while lysosomes did not play a key role. By using dominant-negative mutants and small interfering RNAs, we identified that USP19-mediated secretion of misfolded TDP-43 is modulated by key factors involved in cellular trafficking and secretion pathways, such as ATG7, the ESCRT-O HGS/HRS, the Rab GTPases RAB11A, RAB8A, and RAB27A, and the v-SNARE VAMP7. We also confirmed the crucial role of the DNAJC5/CSPα cochaperone. Overall, this study provides new insights into how cells manage the secretion of misfolded TDP-43 proteins and potentially opens new avenues for therapeutic interventions in ALS and related disorders.
© 2025. The Author(s).
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Biochemistry and Molecular biology
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Preprint on BioRxiv : the Preprint Server for Biology on 30 January 2025 by Parlatan, U., Boudreau, L., et al.
Extracellular vesicles (EV) are becoming crucial tools in liquid biopsy, diagnostics, and therapeutic applications, yet their nanoscale characterization remains challenging. In this context, the detection of drug-induced liver injury, i . e ., hepatotoxicity, through extracellular vesicle molecular content remains an unexplored frontier. To this end, we present a label-free surface-enhanced Raman (SERS) spectroscopy approach, which provides rapid EV content analysis under ten minutes and requires only 1.3 microliters of sample. Using hepatic cultures as a model, our platform captures distinct and reproducible EV molecular changes in response to acetaminophen-induced hepatoxicity. Our platform achieves exceptional accuracy with root mean squared error values as low as 3.80%, establishing strong correlations between EV spectra and conventional toxicity biomarkers. Unlike previous EV-SERS studies limited to vesicle identification and disease markers, this approach reveals EV drug-response signatures strongly correlated with conventional toxicity markers. These findings establish EVs as dynamic reporters of cellular drug responses and demonstrate use of SERS-based EV detection of hepatotoxicity.
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In PLoS Pathogens on 1 November 2024 by Marougka, K., Judith, D., et al.
Human respiratory syncytial virus (RSV) is an enveloped RNA virus and the leading viral agent responsible for severe pediatric respiratory infections worldwide. Identification of cellular factors able to restrict viral infection is one of the key strategies used to design new drugs against infection. Here, we report for the first time that the cellular protein BST2/Tetherin (a widely known host antiviral molecule) behaves as a restriction factor of RSV infection. We showed that BST2 silencing resulted in a significant rise in viral production during multi-cycle infection, suggesting an inhibitory role during the late steps of RSV's multiplication cycle. Conversely, BST2 overexpression resulted in the decrease of the viral production. Furthermore, BST2 was found associated with envelope proteins and co-localized with viral filaments, suggesting that BST2 tethers RSV particles. Interestingly, RSV naturally downregulates cell surface and global BST2 expression, possibly through a mechanism dependent on ubiquitin. RSV's ability to enhance BST2 degradation was also validated in a model of differentiated cells infected by RSV. Additionally, we found that a virus deleted of NS1 is unable to downregulate BST2 and is significantly more susceptible to BST2 restriction compared to the wild type virus. Moreover, NS1 and BST2 interact in a co- immunoprecipitation experiment. Overall, our data support a model in which BST2 is a restriction factor against RSV infection and that the virus counteracts this effect by limiting the cellular factor's expression through a mechanism involving the viral protein NS1.
Copyright: © 2024 Marougka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Immunology and Microbiology
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In Journal of Extracellular Vesicles on 1 November 2024 by Viola, M., Bebelman, M. P., et al.
Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in the heart under homeostatic and pathological conditions, such as myocardial infarction (MI). However, the basic mechanisms driving cardiomyocyte-derived EV (CM-EV) production following stress are poorly understood. In this study, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that express NanoLuc-tetraspanin reporters. These modified hiPSC-CMs allow for quantification of tetraspanin-positive CM-EV secretion from small numbers of cells without the need for time-consuming EV isolation techniques. We subjected these cells to a panel of small molecules to study their effect on CM-EV biogenesis and secretion under basal and stress-associated conditions. We observed that EV biogenesis is context-dependent in hiPSC-CMs. Nutrient starvation decreases CM-EV secretion while hypoxia increases the production of CM-EVs in a nSmase2-dependent manner. Moreover, the inflammatory cytokine TNF-α increased CM-EV secretion through a process involving NLRP3 inflammasome activation and mTOR signalling. Here, we detailed for the first time the regulatory mechanisms of EV biogenesis in hiPSC-CMs upon MI-associated stressors.
© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.
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Homo sapiens (Human)
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Stem Cells and Developmental Biology
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In Int J Mol Sci on 25 July 2024 by Saari, H., Marttila, H., et al.
Fig.2.D
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Homo sapiens (Human)
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...
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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.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 25 July 2024 by Saari, H., Marttila, H., et al.
Fig.3.B
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Homo sapiens (Human)
EVs are enriched from the platelet supernatant and separated from ApoB-positive lipoproteins. (A) Representative SDS-PAGE gel and (B) Western blot with EV markers CD9 and CD63 and ApoB of all the runs from single platelet supernatants (PSs). The peak fraction (Fr3) was used to represent the purif...
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EVs are enriched from the platelet supernatant and separated from ApoB-positive lipoproteins. (A) Representative SDS-PAGE gel and (B) Western blot with EV markers CD9 and CD63 and ApoB of all the runs from single platelet supernatants (PSs). The peak fraction (Fr3) was used to represent the purified EVs from each sample. The gels were loaded using a normalized protein mass of 5 µg per well. (C) Assessment of EV markers CD9 and CD63 and (D) ApoB intensities from the Western blot assays in flowthrough (FT) versus Fr3. The results are shown as box and whisker plots, with marker intensities normalized against the PS. Statistical significance was tested using a two-way ANOVA with Fisher’s LSD post hoc test and denoted by an asterisk: * p < 0.05 or ** p < 0.01 or **** p < 0.0001 (n = 6 for each sample type). Only significant differences due to the fraction type are denoted; no significant differences were found due to sample type.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Commun Biol on 6 May 2024 by Fujita, Y., Kadota, T., et al.
Fig.1.E
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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...
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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.
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Collected and cropped from Commun Biol by CiteAb, provided under a CC-BY license
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In iScience on 18 August 2023 by Bebelman, M. P., Setiawan, I. M., et al.
Fig.2.E
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US28-containing MVBs fuse with the plasma membrane to release exosomal US28(A) Schematic representation of US28-pHluorin-based visualization of MVB-plasma membrane fusion: the pH-sensitive pHluorin is quenched when facing the acidic lumen of the MVB. Upon fusion, the low luminal pH of the MVB is ...
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US28-containing MVBs fuse with the plasma membrane to release exosomal US28(A) Schematic representation of US28-pHluorin-based visualization of MVB-plasma membrane fusion: the pH-sensitive pHluorin is quenched when facing the acidic lumen of the MVB. Upon fusion, the low luminal pH of the MVB is immediately neutralized, resulting in a sudden increase in fluorescence intensity.(B) TIRF imaging (heatmap) of a US28-pHluorin-expressing HeLa cell revealing the sudden appearance of multiple bright fluorescent spots at the plasma membrane. Stills corresponding to Video S1. New spots are highlighted by white arrows. N, nucleus. Dotted and dashed lines outline the nucleus and cell border, respectively. Scale bar: 10 μm.(C) Time-lapse imaging (heat maps) of a single fusion event of US28-pHluorin.(D) Fluorescence signal duration of individual CD63-pHluorin (n = 72, mean = 22 s), VAMP2-pHluorin (n = 79, mean 1.8 s), and US28-pHluorin (n = 76, mean = 23 s) fusion events. Graph depicts pooled data from 8 cells acquired in 2 independent experiments.(E) Western blot for HA-US28 (anti-HA antibody), the sEV marker protein CD63, and β-actin on cell lysates and the sEV pellet isolated by ultracentrifugation from equal numbers of U251 cells or doxycycline-induced iHA-US28-U251 cells. Representative of two independent experiments.
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Collected and cropped from iScience by CiteAb, provided under a CC-BY license
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In mSphere on 22 June 2023 by Schuler, B., Sladek, M., et al.
Fig.2.A
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ICC-IF
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CCV expansion is attenuated in ORP1-null HeLa cells. (A) Representative images of wild-type (WT) and ORP1-null HeLa cells infected with mCherry-expressing C. burnetii for 6 days, then immunofluorescence stained with anti-CD63 antibodies and DAPI. White arrows indicate CCVs. Scale bars, 10 μm. (B)...
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CCV expansion is attenuated in ORP1-null HeLa cells. (A) Representative images of wild-type (WT) and ORP1-null HeLa cells infected with mCherry-expressing C. burnetii for 6 days, then immunofluorescence stained with anti-CD63 antibodies and DAPI. White arrows indicate CCVs. Scale bars, 10 μm. (B) CCV areas in WT and ORP1-null cells after 4 days of infection with mCherry-expressing C. burnetii. WT CCV mean area was 69.65 pixels, and ORP1-null CCV mean area was 51.55 pixels. (C) CCV areas in WT and ORP1-null HeLa cells after 6 days of infection with mCherry-expressing C. burnetii. CCV mean area in WT cells was 101.9 pixels, and ORP1-null CCV mean area was 58.34 pixels. Data shown are means ± standard errors of the mean (SEM) of at least 20 cells per condition in each of three independent experiments as determined by unpaired Student's t test. *, P < 0.05; ****, P < 0.0001.
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Collected and cropped from mSphere by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.
Fig.6.F
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.
Fig.1.C
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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...
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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)
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.
Fig.6.A
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
Image 1 of 70
In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.
Fig.6.B
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WB
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
Image 1 of 70
In J Extracell Vesicles on 1 March 2023 by Marie, P. P., Fan, S. J., et al.
Fig.6.D
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In Blood Cancer Discov on 6 January 2023 by Gargiulo, E., Viry, E., et al.
Fig.1.M
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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...
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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.
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Collected and cropped from Blood Cancer Discov by CiteAb, provided under a CC-BY license
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In Sci Rep on 22 December 2022 by Petit, I., Levy, A., et al.
Fig.1.A
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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...
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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.
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Collected and cropped from Sci Rep by CiteAb, provided under a CC-BY license
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In Inflamm Regen on 4 September 2022 by Kobayashi, M., Fujiwara, K., et al.
Fig.2.C
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TGF-β increases the release of EVs from oral cancer cells. a, b Nanoparticle tracking analysis of EVs from the conditioned medium of HSC-4 cells treated without (a Ctrl-EVs) or with TGF-β (b Tβ-EVs) using the NanoSight system. The inset: NanoSight capture images show particles isolated from the s...
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TGF-β increases the release of EVs from oral cancer cells. a, b Nanoparticle tracking analysis of EVs from the conditioned medium of HSC-4 cells treated without (a Ctrl-EVs) or with TGF-β (b Tβ-EVs) using the NanoSight system. The inset: NanoSight capture images show particles isolated from the same volume of each conditioned medium. c Immunoblotting analysis of total cell lysate of HSC-4 cells stimulated without (Ctrl) or with 3 ng/ml TGF-β (TGF-β) for 72 h, and the respective EV fraction (Ctrl-EVs and Tβ-EVs). Samples derived from the same number of particles were loaded onto each lane. Antibodies against EV markers (Alix, TSG101, and CD63) and Golgi apparatus marker (GORASP1) were used for analysis. Cropped blot images are shown. See Supplementary Information for the original full-length blot images. The intensity of each band was then quantified. The relative value was normalized to that of the cell lysate and is shown as ratio
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Collected and cropped from Inflamm Regen by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.
Fig.5.B
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.
Fig.2.B
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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...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.
Fig.1.F
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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‐...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
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In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.
Fig.1.A
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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‐...
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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
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
Image 1 of 70
In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.
Fig.8.C
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KIT‐EV immunocapture of LAD2 huMC EVs and KIT‐EV recovery from spiked plasma. (A) Cytokine‐starved LAD2 huMCs were treated or not with stem cell factor (SCF; 100 ng/ml) for 2 h in HEPES‐BSA buffer. Cell lysates (30 μg) were analyzed by immunoblotting with the indicated antibodies. (B) EVs release...
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KIT‐EV immunocapture of LAD2 huMC EVs and KIT‐EV recovery from spiked plasma. (A) Cytokine‐starved LAD2 huMCs were treated or not with stem cell factor (SCF; 100 ng/ml) for 2 h in HEPES‐BSA buffer. Cell lysates (30 μg) were analyzed by immunoblotting with the indicated antibodies. (B) EVs released into the supernatant by 18 × 106 LAD2 cells treated or not with SCF for 2 h were sedimented by differential ultracentrifugation into P15 and P120 EVs. KIT‐EV immunocapture was performed (KIT antibody AF332) from all sedimented LAD2 P15 and P120 EVs. The total bead elution was loaded and analyzed by Western blotting with the indicated antibodies. Input EV lysates (5 × 108) were included. Representative blots are shown. (C) HMC‐1.1 cell line‐derived EVs (1.2 × 1010 EVs) were incubated or not in human normal plasma and captured with a KIT antibody (AF332) or by CD9 antibody‐conjugated beads. A goat HA antibody served as a negative isotype control. Representative blots are shown. sKIT, soluble KIT that is present in plasma. Dashed lines imply lanes from the same blot that were not loaded contiguously
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Collected and cropped from J Extracell Vesicles by CiteAb, provided under a CC-BY license
Image 1 of 70
In PLoS Genet on 1 June 2022 by Oe, Y., Kakuda, K., et al.
Fig.3.B
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PACSIN1 depletion causes amphisome accumulation.(A) WT or PACSIN1 KO HeLa cells were cultured for 2 h in growth medium or EBSS, or for 6 h with 125 nM Baf.A1 in growth medium. After fixation, cells were analyzed by electron microscopy. Autophagosomes are indicated by arrowheads, and amphisomes ar...
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PACSIN1 depletion causes amphisome accumulation.(A) WT or PACSIN1 KO HeLa cells were cultured for 2 h in growth medium or EBSS, or for 6 h with 125 nM Baf.A1 in growth medium. After fixation, cells were analyzed by electron microscopy. Autophagosomes are indicated by arrowheads, and amphisomes are indicated by arrows. Scale bars, 500 nm. (B) WT or PACSIN1 KO HeLa cells were cultured for 2 h in growth medium with or without 10 μg/mL E-64-d and pepstatin A. After fixation, cells were co-immunostained with anti-LC3 and anti-CD63 antibodies. Scale bars, 20 μm. (C) The co-localization rate of LC3 with CD63 was quantified using CQ1 software, mean ± s.e.m. More than 200 cells were analyzed per condition in each experiment (n = 5). n.s.; not significant, **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).
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Collected and cropped from PLoS Genet by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 4 April 2022 by Lattekivi, F., Guljavina, I., et al.
Fig.1.C
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Purification and confirmation of blood serum EVs. (A) Elution profile of differentially centrifuged and qEV original 70 nm size-exclusion chromatography (SEC) purification of blood serum EVs in across 500 μL fractions. Consecutively collected fractions are presented on the x-axis, while bars repr...
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Purification and confirmation of blood serum EVs. (A) Elution profile of differentially centrifuged and qEV original 70 nm size-exclusion chromatography (SEC) purification of blood serum EVs in across 500 μL fractions. Consecutively collected fractions are presented on the x-axis, while bars represent the nanoparticle concentration (particles/mL) per fraction, and the blue line marks protein concentration (mg/mL) across fractions. (B) Western blotting analysis for relative abundance of EV-specific surface markers (CD63, CD9, and CD81) and ApoA1 as the major component of HDL particles in the nanoparticle-rich SEC fractions. Equal volumes of concentrated EV samples were used as the input in case of the tetraspanins, while the input was diluted 10-fold for ApoA1. (C) Comparative Western blotting of EV-specific surface markers and ApoA1 in the purified EV samples in contrast to unprocessed blood serum with total protein concentration adjusted to be equal for both samples for each target protein. (D,E) Transmission electron microscopy imaging of purified EV samples. Black arrows highlight spherical particles with size and morphological characteristics inherent to EVs.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 4 April 2022 by Lattekivi, F., Guljavina, I., et al.
Fig.1.B
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Purification and confirmation of blood serum EVs. (A) Elution profile of differentially centrifuged and qEV original 70 nm size-exclusion chromatography (SEC) purification of blood serum EVs in across 500 μL fractions. Consecutively collected fractions are presented on the x-axis, while bars repr...
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Purification and confirmation of blood serum EVs. (A) Elution profile of differentially centrifuged and qEV original 70 nm size-exclusion chromatography (SEC) purification of blood serum EVs in across 500 μL fractions. Consecutively collected fractions are presented on the x-axis, while bars represent the nanoparticle concentration (particles/mL) per fraction, and the blue line marks protein concentration (mg/mL) across fractions. (B) Western blotting analysis for relative abundance of EV-specific surface markers (CD63, CD9, and CD81) and ApoA1 as the major component of HDL particles in the nanoparticle-rich SEC fractions. Equal volumes of concentrated EV samples were used as the input in case of the tetraspanins, while the input was diluted 10-fold for ApoA1. (C) Comparative Western blotting of EV-specific surface markers and ApoA1 in the purified EV samples in contrast to unprocessed blood serum with total protein concentration adjusted to be equal for both samples for each target protein. (D,E) Transmission electron microscopy imaging of purified EV samples. Black arrows highlight spherical particles with size and morphological characteristics inherent to EVs.
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Micromachines (Basel) on 21 November 2021 by Shimasaki, T., Yamamoto, S., et al.
Fig.6.B
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Validation of the ability of extracellular vesicles (EV) to pass through the filter according to pore size. (a) Scheme of the evaluation method for EVs secreted from PANC-1 cells. (b) Evaluation of the passing ability of extracellular vesicles (EVs). The same number of EVs from PANC-1 cells were ...
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Validation of the ability of extracellular vesicles (EV) to pass through the filter according to pore size. (a) Scheme of the evaluation method for EVs secreted from PANC-1 cells. (b) Evaluation of the passing ability of extracellular vesicles (EVs). The same number of EVs from PANC-1 cells were seeded on one side (left side) of the horizontal-type co-culture plate (HTCP). The number of exosomes passing through the filter on the vessel’s right side were measured using Western blot. The 0.03 μm filter blocked the transfer of the EVs. The absence of the exosome on the adjacent vessel is confirmed by the Western blotting method in experiments with pore size 0.03 μm. Markers of exosomes (CD9, CD63, CD81, Flotilin-1) were not detected at 72 h.
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Collected and cropped from Micromachines (Basel) by CiteAb, provided under a CC-BY license
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In Cancers (Basel) on 26 October 2021 by Chen, Y. Y., Dagg, R., et al.
Fig.4.M
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Purification of the exosomes by iodixanol density gradient separation: (a) schema of the iodixanol density gradient separation method (upper panel) and the density distribution in each fraction collected (lower panel). (b) Representative Western blot of exosome-specific proteins CD63, CD81, and S...
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Purification of the exosomes by iodixanol density gradient separation: (a) schema of the iodixanol density gradient separation method (upper panel) and the density distribution in each fraction collected (lower panel). (b) Representative Western blot of exosome-specific proteins CD63, CD81, and Syntenin in the iodixanol fractions. The positions of the protein size standards (M) are shown. (c) Quantitation (n = 3) of the Western blots presented as the percentage of the total combined signal for each fraction. (d) Representative C-Circle assay (CCA) dot blot of the iodixanol fractions, with the sample-specific background (Background) checked by omitting the Phi29 DNA polymerase from the CCA. An overexposed CCA dot blot is presented in Figure S3 to allow for better visualization of the Background signal. (e) Quantitation (n = 3) of the CCA presented as the percentage of the total (combined) CCA signal from all fractions. The major CCA peak was in F2, which contained 52% (95% CI = 10%) of the total CCA signal from all fractions. All error bars indicate the SEM.
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Collected and cropped from Cancers (Basel) by CiteAb, provided under a CC-BY license
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In Cancers (Basel) on 26 October 2021 by Chen, Y. Y., Dagg, R., et al.
Fig.5.B
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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...
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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.
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Collected and cropped from Cancers (Basel) by CiteAb, provided under a CC-BY license
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In iScience on 20 August 2021 by Bouchareychas, L., Duong, P., et al.
Fig.4.B
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Assessment of plasma EVs isolated from subjects with diabetes and atherosclerosis(A) Details of clinical data of the cohort used in this study.(B) Analyses of 9 individual fractions of plasma EVs separated by C-DGUC method. An equal volume (37.5 μL) of fraction samples was loaded for Western blot...
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Assessment of plasma EVs isolated from subjects with diabetes and atherosclerosis(A) Details of clinical data of the cohort used in this study.(B) Analyses of 9 individual fractions of plasma EVs separated by C-DGUC method. An equal volume (37.5 μL) of fraction samples was loaded for Western blot analysis.(C–E) (C) Particle diameter distribution profiles for fraction 8 estimated by NTA. Particle concentrations (D) and size (E) were estimated by NTA using NanoSight.(F) Protein concentration was measured by qubit assay. Statistical analysis was performed using one-way ANOVA and Sidak's multiple comparisons post-test. ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001. Data are represented as mean ± SEM.
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Collected and cropped from iScience by CiteAb, provided under a CC-BY license
Image 1 of 70