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2016年5月10日 星期二

Cell-Surface Vimentin Monoclonal Antibody

Vimentin, a 57kDa type III intermediate filament, is responsible for architecture of cytoplasm.1 As a popular intracellular epithelial mesenchymal transition (EMT) marker, overexpression of vimentin in cancer cells was known for its high correlation with cancer progression. Apart from secretion under some circumstances, vimentin can also be recruited to the cell surface, known as cell surface vimentin (CSV), to participate in cell adhesion, migration and cellular signaling.2

The significance of CSV is related to its involvement in autoimmune disorders, viral infection and cancer. CSV expression on activated marcophages, platelets and apoptotic T lymphocytes evidenced its connection to human neutrophil spontaneous apoptosis further supports its association with inflammatory diseases.3 Being a putative anti-viral drug target, CSV also takes parts in viral multiplication by directly facilitate internalization of virion.4 Recently, breakthroughs on identifying CSV as cancer specific EMT marker, in association with metastatic phenotypes in aggressive cancer and sarcoma, has made an impact on cancer research community. Because unlike intracellular vimentin, the cell surface detection of CSV enables the isolation of cancer cells.5,6,7

Abnova obtains worldwide exclusive license of the patented CSV monoclonal antibody, clone 84-1, from MD Anderson Cancer Center, United States. This antibody has been validated for its intended use in majorities of cancer cell lines by both immunofluoresence and flow cytometry.8 As a sole provider for this powerful tool, Abnova hopes to propel the investigations on cancer research with specificity and broad applicability.

CSV monoclonal antibody, clone 84-1
H00007431-M08


CSV Expression in Different Cell Lines8
Cell Line CSV Cell Line CSV Cell Line CSV
Breast   Bone   Liver  
  MCF-7 (H) +   OS-25 (H) ++   AMC14 (M) ++
  SKBR3 (H) +   HOS (H) ++ Colon  
  MDA-MB-231 (H) +   MG-263 (H) ++   DLD-1 (H) ++
  MDA-MB-453 (H) +   LM7 (H) +   GEO (H) ++
  MDA-MB-458 (H) ++   SAOS-2 (H) +   OS-187 (H) ++
  4T1 (M) +   OS25 (H) +   SW620 (H) +
Brain     OS-O (H) ++   SW480 (H) +
  SKNAS (H) ++   OS-D (H) +   HCT-116 (H) +
  SKNBE2 (H) +++   U2OS (H) +   HT-29 (H) ++
  NGP (H) +   CCH-OSD (H) +   Caco-2 (H) +
  SH-SY5Y (H) ++   K7 (M) ++   CT-26 (M) +
  LAN5 (H) ++   K7M2 (M) +++ Pancreas  
  KCN (H) +   DUNN (M) +   PANC-1 (H) ++
  DBT (M) +   LM8 (M) +++   MiaPACA-2 (H) +
  U251 (H) +   OST-DL-391 + Other  
Bladder     OST-DL-393 +   FBL3 (M) +++
  RT4V6 (H) +   OST-DL-396a ++   SCCVII (M) +
  T24 (H) ++        

Flow Cytometry Using CSV monoclonal antibody, clone 84-1 (H00007431-M08)
CSV Expression in Cancer Cell Lines
Immunological assessment of CSV in various cancer cell lines and normal cell lines using flow cytometry in which only cancer cells, including GEO (colorectal cancer), MDA-MB-231 (breast cancer) and PANC-1 (pancreatic cancer) can be detected by CSV, 84-1.6
CSV Expression in Sarcoma Cell Lines
Immunological assessment of CSV in various cancer cell lines and normal cell lines using flow cytometry in which only cancer cells, including SKNBE-2 (neuroblastoma), RH-41 (rhabdomyosarcoma) and LM7 (osteosarcoma) can be detected by CSV, 84-1.5
Sorting of Hepatocellular Carcinoma Cells (HCC)
Stem-cell liked cancer cell populations, csVim+ CD133- and csVim- CD133+, were isolated and sorted into sub-G1, G1, S and G2 phases from primary HCC using CSV, 84-1.7

Immunofluorescence Using CSV monoclonal antibody, clone 84-1 (H00007431-M08)
Analysis of CSV-specific 84-1 monoclonal antibody
Cell surface staining of CSV in LM7 (osteosarcoma) cancer cell line using confocal microscopy. Cells were stained for CSV (green), WGA (red) and nucleus DRAQ5 (blue). CSV monoclonal antibody 84-1 co-localized with WGA indicated cell surface vimentin.5
Detection of CSV in HPC-1-derived Spheres on Geltrex-coated Wells
Vimentin (green) was detected at the periphery of 3-dimensional HPC-derived sphere model. Since cells at periphery were more aggressive, the invasivephenotype correlated with increase nuclear accumulation of β-catenin (red).6
Detection of CSV on Human Colorectal Cancer Cells
Circulating tumor cells derived from colorectal cancer patients were stained for nucelus (blue), cell surface vimentin (green) and specific EMT biomarkers (red), including FOXC2, TWIST-2, SNAIL, SLUG, EpCAM and E-cadherin.6

References
1. Mitra, A. and Li, S. (2014). J Cancer Prev Curr Res. DOI: 10.15406/jcpcr.2014.01.00014
2. Satelli, A. and Li, S. (2011). Cell Mol Life Sci. DOI: 10.1007/s00018-011-0735-1
3. Moisan, E. and Girard, D. (2005). JLB. DOI:10.1189/jlb.0405190
4. Yu, YT., et. al. (2016). Journal of Biomedical Science. DOI: 10.1186/s12929-016-0234-7
5. Satelli A., et al. (2014). Cancer Research. DOI:10.1158/0008-5472.CAN-13-1739.
6. Satelli A., et al. (2014). Clinical Cancer Research. DOI:10.1158/1078-0432.CCR-14-0894.
7. Mitra, A., et al. (2015). IJC. DOI:10.1002/ijc.29382.
8. Satelli A. (2014). WIPO. WO 2014/138183 A1.

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2015年8月5日 星期三

Cytoskeleton

New Discoveries on Cytoskeletons
  • Self-organization of actin cytoskeleton, the dynamic of radial fibers-transverse fibers system, takes crucial role in developing the left-right asymmetry in tissues and embryos. (Nature Cell Biology,2015)
  • Apical and basolateral spectrin cytoskeletons, potential mechano-sensors, regulate Hippo signaling pathway in response to human epithelial cell density. (EMBO,2015)
  • Cytoskeleton-associated proteins, CRMP2/DPYSL2, TPM2, TCP1ε and 14.3.3γ, can be potential biomarkers for small-intestinal neuroendocrine tumors invasion. (Molecular and Cellular Endocrinology,2015)

Cytoskeleton Compositions
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Proteins Involved in Cytoskeletons
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Actin Polymerization
Protein Function/Activity
Cofilin Dissociation from (-) end
Severin Severing, capping (+) end, control of length of actin filaments; essential for branch formation
Gelsolin Severing, capping (+) end, control of length of actin filaments; essential for branch formation
CapZ capping protein Capping (+) end, stabilize F-actin prevent disassembly during muscle contraction
Tropomodulin Capping (+) end, stabilize F-actin prevent disassembly during muscle contraction
ARPC 2/3 Capping (-) end, side binding and nucleation
Classes of Intermediate Filaments
Protein Function/Activity
I. Acidic cytokeratins Mechanical strength
II. Basic cytokeratins Mechanical strength
III. Vimentin
III. Desmin
III. GFAP
Maintenance of cell shape
Structural support for contractile machinery
Maintenance of cell shape
IV. Neurofilament
(NEFL,NEFM,NEFH)
Axon strength; Determines axon size
V. Nuclear Lamins
(Lamin A, Lamin B)
Form nuclear scaffold and shape the nucleus
VI. Nestin Unknown
Modulation of Microtubule Dynamics
Protein Function
MAP1 Assembles and stabilizes microtubules
MAP2 Assembles and cross-links microtubules to one another and intermediate filaments
MAP4 Stabilizes microtubules
MAPT Assembles and stabilizes and cross-links microtubules
RSN/CLIP1 Cross-links microtubules to chromosomes and endosomes
Katanin Microtubule severing
STMN1/OP18 Binds to tubulin dimers

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CTNNA1 polyclonal antibody
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Immunohistochemistry staining of paraffin embedded human prostate using CTNNA1 polyclonal antibody (Cat# PAB7024) at 3.8μg/mL.

References
Tee, Y.H., et. al (2015). Nature Cell Biology. DOI: 10.1038/ncb3137
Fletcher, G.C., et.al.(2015). EMBO Journal. DOI 10.15252/embj.201489642
Couderc, C., et.al. (2015). Molecular and Cellular Endocrinology. DOI:10.1016/j.mce.2014.09.006

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2015年7月14日 星期二

Hematopoiesis

Abnova July E-Newsletter
New Discoveries on Hematopoiesis
  • Hematopoietic stem cell (HSC) is generated from the hemogenic endothelium (HE), where HE and arterial vascular endothelium (VE) was found to represent separate lineages, on the basis of differential expression of CD73 and CD184. (Nature Cell Biology, 2015)
  • HSC regenerative capacity was increased through the inactivation of a histone deacetylase, SIRT7. The interplay of SIRT7 and NRF1 deregulates an unfolded protein response (UPRmt)-mediated metabolic checkpoint which was defined as a reversible contributing factor for HSC aging. (Science, 2015)
  • TET1, the founding member of TET family, was reported as an epigenetic guardian for lymphomagenesis and the TET1 deficiency contributed to predisposed development of B cell malignancy. (Nature Immunology, 2015)

Blood Cell Specific Markers
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Cell Type Markers (Human) Markers (Mouse)
Hematopoietic Stem Cells Lin-, CD34+, CD38-, FLT3+, THY1+ Lin-, Slamf1 (Cd150)+, Kit+, Casp3 (Sca-1)+
Lin- MS4A1 (CD20), GYPA (CD235a), CD3, NCAM1 (CD56) Ptprc (Cd45ra), Itgam (Cd11b), Cd2, Cd3, Cd4, Cd5, Cd8, Ly6g, Klrb1c, Ly76
Common Lymphoid Progenitor (CLP) MME (CD10)+, CD34+, IL7R (CD127)+ Cd34+, Il7r (Cd127)+
Common Myelold Progenitor (CMP) CD34+, IL3RA (CD123)+, FLT3+ Cd34+, Il3ra (Cd123)+, Casp3 (Sca-1)-
Granulocyte/Macrophage Progenitor (GMP) CD34+, CD38+, IL3RA (CD123)+ Cd34+, Cd38+, Il3ra (Cd123)+
Megakaryocyte/Erythroid Progenitor (MEP) CD38+, PTPRC (CD45RA)- Cd38+, Ptprc (Cd45ra)-
Megakaryocytes ITGA2B (CD41)+, MT2A (CD42)+, MPL (TPOR)+ Itga2b (Cd41)+, Mt2a (Cd42)+, Mpl (Tpor)+
Monocytes CD14+, CXCR1+, ITGAM (CD11b)+, CD33 (Siglec-3)+ Cd14+, Cxcr1+, Itgam (Cd11b)+, Cd33 (Siglec-3)+
Lymphocytes LY9+, SLAMF7+ Ly9+, Slamf7+
Natural Killer Cells (NK Cells) NCAM1 (CD56)+, CD16+ Ncr1 (Cd335) +, Fcgr3 (CD16)+
B Cells CD19+, MS4A1 (CD20)+ Ptprc (Cd45ra)+, Cd19+, Cd22+
T Cells CD2+, CD3+, CD4+, CD8+ Cd2+, Cd3+, Cd4+, Cd8+
Granulocytes CCR3+, ITGAM (CD11b)+, ANPEP (CD13)+, CD16+, ITGB2 (CD18)+ Ccr3+, Itgam (Cd11b)+, Anpep (Cd13)+, Fcgr3 (Cd16)+, Itgb2 (Cd18)+
Basophils ANPEP (CD13)+, CD164+, IL3RA (CD123)+, LAMP1 (CD107a)+ Anpep+, Cd164+, Il3ra (CD123)+, Lamp1 (Cd107a)+
Neutrophils CD66+, SELL (CD62L)+, MPO+ Ceacam1 (Cd66)+ , Sell+, Mpo+
Eosinophils CD44+, CD69+, EMR1+ Cd44+, Cd69+, Emr1+
Erythrocytes GYPA (CD235a)+, CD59+ Gypa (Cd235a)+, Cd59a+
Dendritic Cells IL3RA (CD123)+, CD80+, CD83+, CD86+, HLA-DRA+ Il3ra (Cd123)+, Cd80+, Cd83+, Cd86+
Macrophages CD14+, CD163+, CD36+, CD68+ Cd14+, Cd163+, Cd36+, Cd68+
Platelets ITGA2B (CD41)+, ITGB3 (CD61)+, PEAR1+ Itga2b (Cd41)+, Itgb3 (Cd61)+, Pear1+

Hematopoiesis
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References
Ditadi A, et.al. (2015). Nature Cell Biology. DOI: 10.1038/ncb3161.
Mohrin M, et. al. (2015) Science. DOI: 10.1126/science.1258849.
Rasmussen KD, et. al. (2015) Nature Immunology. DOI: 10.1038/ni.3176

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2015年5月11日 星期一

Kiss of Death - Ubiquitylation

Abnova May E-Newsletter
New Discoveries on Ubiquitylation
  • Elongation of ubiquitin chain, regardless of the linkage, can form polyubiquitin fibrils to initiate autophagy pathway. Incapacity of autophagy can cause cytotoxic accumulation of ubiquitin-positive aggregates leading to neurodegenerative diseases. (Nature communication,2015)
  • Highly expressed in human breast and lung cancers, a new deubiquitinating enzyme, USP36, was identified to regulate c-Myc oncoprotein stability in nucleolus. (PNAS,2015)
  • Itch, an E3 HECT ubiquitin ligase, inhibits MAPK p38α activation through ubiquitylation can be exploited therapeutically to prevent chronic skin inflammation. (Science,2015)

Tumor Related Misregulated Proteins in Ubiquitin Modification
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Tumor Type Deregulated Protein Substrate Modification
Non-Small-Cell Lung Cancer MDM2 (39) p53/TP53 (151) Polyubiquitylation
HAUSP/USP7 (8) MDM2(39)
p53/TP53 (151)
De-ubiquitylation
Renal Cell Carcinoma VHL (16) HIF/HIF1/HIF1α (23) De-ubiquitylation
Colorectal Cancer APC (69) Cyclin B (31)
Securin
Polyubiquitylation
Lymphoma KITLG/SCF (25) CDKN1B/p27 (51) Polyubiquitylation
Fanconi Anaemia-Related Cancer ATAD3A/FANCL (9) FANCD2 (9) Monoubiquitylation
MALT Lymphomas IAP2 BCL10 (15) Polyubiquitylation
Lymphoma, AML, Gastric Carcinoma CBL (33) RTKs Multiple Monoubiquitylation
Cervical Cancer UBE3A/E6-AP (11) p53/TP53 (151) Polyubiquitylation
Breast Cancer BRCA1 (75) Unknown Unknown
CBL (33) EGFR (117) De-ubiquitylation
Ovarian Cancer F-box protein Cyclin E/CCNE1 (43) De-ubiquitylation
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Human ovary cancer (FFPE) stained with CCNE1/CEN19p FISH Probe. Human ovary cancer showed no CCNE1 gene amplification.

References
Morimoto, D., et.al. (2015). Nature communications. DOI: 10.1038/ncomms7116
Sun, X.-X., et.al. (2015). PNAS. DOI: 10.1073/pnas.1411713112
Theivanthiran, B., et.al. (2015). Science. DOI: 10.1126/scisignal.2005903
Yumimoto, K., et.al. (2015) JCI. DOI: 10.172/JCI78782
Noske, A., et.al. (2015). Experiment and Molecular Pathology.
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