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- Liang, Z P, et al.
(författare)
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Electrochemical study of the XNA on Gold (TM) microarray
- 2004
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Ingår i: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 20:2, s. 211-216
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Tidskriftsartikel (refereegranskat)abstract
- A novel electrode array was developed based on the XNA on Gold(TM) microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold(TM) coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold(TM) electrode. The electrochemical reaction of K4Fe(CN)(6) was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)(6) to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold(TM) can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold(TM) microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis. (C) 2004 Elsevier B.V. All rights reserved.
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| 2. |
- Yang, X. M., et al.
(författare)
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Overexpression of Rac GTPase Activating Protein 1 Contributes to Proliferation of Cancer Cells by Reducing Hippo Signaling to Promote Cytokinesis
- 2018
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Ingår i: Gastroenterology. - : Elsevier BV. - 0016-5085. ; 155:4, s. 1233-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND & AIMS: Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. METHODS: Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and mass spectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. RESULTS: The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10 genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCC cells. CONCLUSIONS: In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation of HCC cells by reducing activation of the Hippo and YAP pathways and promoting cytokinesis in coordination with TPR.
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