The tetraspanin CD9 affords high purity capture of all murine hematopoietic stem cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Shamit Soneji
PROVIDER: E-GEOD-47707 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Prospective isolation is critical to understand the cellular and molecular aspects of stem cell heterogeneity. Here we identify the cell surface antigen CD9 as a novel positive marker that provides a simple alternative for hematopoietic stem cell-isolation at high purity
2013-09-01 | GSE47707 | GEO
Project description:Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. Exogenous expression of human CD9 by CD9-adenoviral transduction led to increases in neointima. CD9 expression significantly augmented PI-3 kinase dependent Akt phosphorylation with concurrent adhesion to extracellular matrix protein fibronectin (FN). Furthermore,enhanced Akt phosphorylation was attenuated by anti-CD9 mAb7 binding. As multiple factors contribute to the regulation of VSMC phenotype, our aim was to establish whether increased CD9 expression would induce changes in the expression of other genes associated with VSMC proliferation and motility. RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data. Analysis of this data showed the regulation of some key genes involved in the modulation of VSMC motility and proliferation. Keywords: vascular smooth muscle cells, vascular injury, tetraspanin, proliferation, neointima, motility, fibronectin (FN), rat aortic smooth muscle (RASM)
2009-11-12 | GSE13594 | GEO
Project description:Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. Exogenous expression of human CD9 by CD9-adenoviral transduction led to increases in neointima. CD9 expression significantly augmented PI-3 kinase dependent Akt phosphorylation with concurrent adhesion to extracellular matrix protein fibronectin (FN). Furthermore,enhanced Akt phosphorylation was attenuated by anti-CD9 mAb7 binding. As multiple factors contribute to the regulation of VSMC phenotype, our aim was to establish whether increased CD9 expression would induce changes in the expression of other genes associated with VSMC proliferation and motility. RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data. Analysis of this data showed the regulation of some key genes involved in the modulation of VSMC motility and proliferation. Experiment Overall Design: RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data.
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Project description:We developed a column-based CD9 antibody-immobilized HPLC immunoaffinity (CD9-HPLC-IAC) technology for EV isolation from a microliter-scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 μL of serum in 30 min with a yield of 8.0×109 EVs. Proteomic analysis showed that the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101 were all identified in EVs. Statistical analysis of EV protein content showed that the top 10 serum proteins in EVs was significantly decreased by using the CD9-HPLC-IAC method compared to the ultracentrifugation (UC) (p=0.001) and size exclusion chromatography (SEC) (p=0.009), and apolipoproteins significantly reduced 4.8-fold compared to the SEC method (p<0.001). The result demonstrated the potential of the CD9-HPLC-IAC method for efficient isolation and proteomic characterization of EVs from a micro-scale volume of serum.
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Project description:Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
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Project description:The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the quantity and type of EVs that are obtained. The aim of this study was therefore to determine the presence of different subpopulations of EVs in plasma and serum. Blood samples were collected from healthy subjects, from which plasma and serum were isolated in parallel. ACD-A or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. We previously developed a method utilising a combination of density cushion and size exclusion chromatography to isolate EVs from plasma with minimal contamination of lipoprotein particles. In the current study, we applied this method to both EDTA-plasma and serum. In addition, EVs were isolated by a combination of density cushion and density gradient or by bead immune capturing (anti-CD63, anti-CD9, and anti-CD81 beads). The subpopulations of EVs were analysed by nanoparticle tracking analysis, Western blot, single particle interferometric reflectance imaging sensing, conventional and nano flow cytometry, magnetic bead enzyme-linked immunosorbent assay, and mass spectrometry.This study shows that a larger number of CD9+ EVs are present in EDTA-plasma compared to ACD-plasma and serum, and the presence of CD41a on theses EVs suggests that they are released from platelets. Furthermore, only a small number of EVs in blood were double positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Together, these findings show that multiple subpopulations of EVs are present in blood including CD9+/CD41a+, CD9+/CD63+/CD41+, CD63+/CD41a+, CD63+/CD9+/CD81−, CD81+/CD9+/CD63−, and CD9+/CD63+/CD81+ and that their presence is dissimilar in EDTA-plasma, ACD-plasma and serum.
2022-05-20 | PXD026863 | Pride
Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration.
2015-02-27 | GSE66336 | GEO
Project description:The tetraspanin family of proteins with four membrane-spanning proteins function in a wide range of physiological processes in higher organisms, including cell migration and proliferation, cell fusion, fertilization and virus infection. Although the recently reported structure of CD81 unveiled the basic architecture of this family for the first time, further structural and functional studies are required in order to understand the mechanistic details of the complicated functions of the tetraspanin-family proteins. In this study, attempts were made to crystallize human CD9, a representative member of the tetraspanin family, and it was demonstrated that the truncation of a variable region in the second long extracellular loop significantly improved crystal growth.
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Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration. Three independent batches were used.
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