Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations
ABSTRACT Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2025-08-01
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| Series: | Journal of Extracellular Vesicles |
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| Online Access: | https://doi.org/10.1002/jev2.70142 |
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| _version_ | 1849222181201903616 |
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| author | Xinming Liu Zubair Ahmed Nizamudeen Christopher J. Hill Christopher Parmenter Kenton P. Arkill Daniel W. Lambert Stuart Hunt |
| author_facet | Xinming Liu Zubair Ahmed Nizamudeen Christopher J. Hill Christopher Parmenter Kenton P. Arkill Daniel W. Lambert Stuart Hunt |
| author_sort | Xinming Liu |
| collection | DOAJ |
| description | ABSTRACT Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC‐derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault‐like particles were visualised in 100k DC pellets by cryo‐transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co‐elution of EV markers and vault particle proteins. Immunocapture of EVs post‐ultracentrifugation (100k DC pellet) showed co‐purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP‐negative. The current study highlights the importance of determining the topology of putative EV‐associated components to determine if they are EV cargo or contaminants that have been co‐purified. |
| format | Article |
| id | doaj-art-cfffd9a2c5f7429e81eb34db1a3dee19 |
| institution | Kabale University |
| issn | 2001-3078 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj-art-cfffd9a2c5f7429e81eb34db1a3dee192025-08-26T07:24:44ZengWileyJournal of Extracellular Vesicles2001-30782025-08-01148n/an/a10.1002/jev2.70142Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle PreparationsXinming Liu0Zubair Ahmed Nizamudeen1Christopher J. Hill2Christopher Parmenter3Kenton P. Arkill4Daniel W. Lambert5Stuart Hunt6School of Clinical Dentistry The University of Sheffield Sheffield UKTranslational Medical Sciences, The University of Nottingham Biodiscovery Institute The University of Nottingham Nottingham UKCryo‐Electron Microscopy Facility The University of Sheffield Sheffield UKNanoscale and Microscale Research Centre The University of Nottingham Nottingham UKTranslational Medical Sciences, The University of Nottingham Biodiscovery Institute The University of Nottingham Nottingham UKSchool of Clinical Dentistry The University of Sheffield Sheffield UKSchool of Clinical Dentistry The University of Sheffield Sheffield UKABSTRACT Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC‐derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault‐like particles were visualised in 100k DC pellets by cryo‐transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co‐elution of EV markers and vault particle proteins. Immunocapture of EVs post‐ultracentrifugation (100k DC pellet) showed co‐purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP‐negative. The current study highlights the importance of determining the topology of putative EV‐associated components to determine if they are EV cargo or contaminants that have been co‐purified.https://doi.org/10.1002/jev2.70142extracellular vesiclesmajor vault proteinvault particlevault RNA |
| spellingShingle | Xinming Liu Zubair Ahmed Nizamudeen Christopher J. Hill Christopher Parmenter Kenton P. Arkill Daniel W. Lambert Stuart Hunt Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations Journal of Extracellular Vesicles extracellular vesicles major vault protein vault particle vault RNA |
| title | Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations |
| title_full | Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations |
| title_fullStr | Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations |
| title_full_unstemmed | Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations |
| title_short | Assessment of Vault Particles in Cancer Cell Line‐Derived Extracellular Vesicle Preparations |
| title_sort | assessment of vault particles in cancer cell line derived extracellular vesicle preparations |
| topic | extracellular vesicles major vault protein vault particle vault RNA |
| url | https://doi.org/10.1002/jev2.70142 |
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