Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Damaraju, Sambasivarao; Zhang, Nancy R.; Li, Nan; Tao, Lidan; Damaraju, Vijaya L.; Dufour, Jennifer; Santos, Cheryl; Sun, Xuejun; Mackey, John R.; Wishart, David S.; Cass, Carol E.; Li, Liang

doi:10.1016/j.ab.2009.08.019

Cited by 2 publications

(4 citation statements)

References 42 publications

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“…MN from OVCAR-5 cells were isolated via sucrose gradient ultracentrifugation, and fractions enriched for MN were washed and submitted for MS (Fig. 4A) ( 19 ). MN enrichment was confirmed with 4′,6-diamidino-2-phenylindole (DAPI) staining and IF (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Methods for isolation and purification of cultured cell MN were adapted from Damaraju et al ( 19 ) with the following modifications: Harvested OVCAR-5 cells were scraped from 15-cm tissue cultures using ice-cold PBS supplemented with protease and phosphatase inhibitors and pelleted by centrifugation at 500 g for 10 min at 4°C.The supernatant was carefully discarded, and the cell pellet was gently resuspended in 1 ml of ice-cold cell lysis buffer obtained from the Active Motif ChIP-IT Express Chromatin Immunoprecipitation Kit supplemented with protease and phosphatase inhibitors provided by the kit (53008, Active Motif). Cells were incubated in lysis buffer on ice for 30 min and then carefully pipetted into a 1-ml Dounce homogenizer.…”

Section: Methodsmentioning

confidence: 99%

“…Supernatant from this step, which contained MN, was kept, and pelleted cell nuclei were discarded. Cell supernatant was loaded to a sucrose gradient (84097-1KG, Sigma-Aldrich) prepared in PBS by using fractions indicated by Damaraju et al ( 19 ). The fraction corresponding to 25% sucrose was collected and mixed with an equal volume of ice-cold PBS.…”

Section: Methodsmentioning

confidence: 99%

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Mechanisms of nuclear content loading to exosomes

et al. 2019

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Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mechanisms of nuclear content loading to exosomes

et al. 2019

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“…A sucrose density gradient was generated as described [35] and the extracts of H1299 cells were loaded to the continuous gradient. After centrifugation, the proteins were collected from different fractions, run on SDS-PAGE, and transferred to membranes.…”

Section: Methodsmentioning

confidence: 99%

Interaction between NBS1 and the mTOR/Rictor/SIN1 Complex through Specific Domains

Wang

Chen

et al. 2013

PLoS ONE

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Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome. The NBS gene product, NBS1 (p95 or nibrin), is a part of the Mre11-Rad50-NBS1 complex. SIN1 is a component of the mTOR/Rictor/SIN1 complex mediating the activation of Akt. Here we show that NBS1 interacted with mTOR, Rictor, and SIN1. The specific domains of mTOR, Rictor, or SIN1 interacted with the internal domain (a.a. 221-402) of NBS1. Sucrose density gradient showed that NBS1 was located in the same fractions as the mTOR/Rictor/SIN1 complex. Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets. Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity. These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221–402 domain and contributes to the activation of Akt activity.

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Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Cited by 2 publications

References 42 publications

Mechanisms of nuclear content loading to exosomes

Mechanisms of nuclear content loading to exosomes

Interaction between NBS1 and the mTOR/Rictor/SIN1 Complex through Specific Domains

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