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Proximity ligation assay

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https://www.readbyqxmd.com/read/29164763/tmem207-hinders-the-tumour-suppressor-function-of-wwox-in-oral-squamous-cell-carcinoma
#1
Katsuaki Bunai, Hiroshi Okubo, Kimika Hano, Keisuke Inoue, Yusuke Kito, Chiemi Saigo, Toshiyuki Shibata, Tamotsu Takeuchi
The WW domain-containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX-mediated regulation of the HIF-1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non-tumorous epithelial tissues...
November 22, 2017: Journal of Cellular and Molecular Medicine
https://www.readbyqxmd.com/read/29157747/multiplex-proximity-ligation-assay-to-identify-potential-prognostic-biomarkers-for-improved-survival-in-locally-advanced-pancreatic-cancer-patients-treated-with-stereotactic-body-radiation-therapy
#2
Avani D Rao, Yufei Liu, Rie von Eyben, Charles C Hsu, Chen Hu, Lauren M Rosati, Arti Parekh, Kendall Ng, Amy Hacker-Prietz, Lei Zheng, Timothy M Pawlik, Daniel A Laheru, Elizabeth M Jaffee, Matthew J Weiss, Dung T Le, Ralph H Hruban, Ana De Jesus-Acosta, Christopher L Wolfgang, Amol K Narang, Daniel T Chang, Albert C Koong, Joseph M Herman
PURPOSE: To explore seromarker levels for associations with outcomes in locally advanced pancreatic cancer (LAPC) patients who received chemotherapy and stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: Serum from LAPC patients in 2 prospective trials of hypofractionated SBRT (5-6.6 Gy × 5) was collected before SBRT. Proximity ligation assay quantified the expression levels of 36 pancreatic cancer-specific candidate seromarkers: Axl, BMP2, CA 125, CA 19-9, CEA, CXCL-1/6/9/10, EGFR, Gas6, Her2, IGF-2, IGFBP-2/3/7, IL-6/6Ra/7/8/12, mesothelin, MMP-1/2/3/7, osteopontin, PDGFRa, PDK1, PF4, RegIV, SPARC, TGF-β, VEGF-A/D, and YKL40...
October 12, 2017: International Journal of Radiation Oncology, Biology, Physics
https://www.readbyqxmd.com/read/29151270/asct2-defined-by-enzyme-mediated-activation-of-radical-sources-enhances-malignancy-of-gd2-plus-small-cell-lung-cancer
#3
Nobutoshi Esaki, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Tsuda, Yuhsuke Ohmi, Robiul H Bhuiyan, Norihiro Kotani, Koichi Honke, Atsushi Enomoto, Masahide Takahashi, Keiko Furukawa, Koichi Furukawa
Ganglioside GD2 is specifically expressed in small cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we performed enzyme-mediated activation of radical sources combined with mass spectrometry in GD2 positive (+) SCLC cells. Consequently, we identified ASC amino-acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2...
November 19, 2017: Cancer Science
https://www.readbyqxmd.com/read/29146920/andy-s-algorithms-new-automated-digital-image-analysis-pipelines-for-fiji
#4
Andrew M K Law, Julia X M Yin, Lesley Castillo, Adelaide I J Young, Catherine Piggin, Samuel Rogers, Catherine Elizabeth Caldon, Andrew Burgess, Ewan K A Millar, Sandra A O'Toole, David Gallego-Ortega, Christopher J Ormandy, Samantha R Oakes
Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays...
November 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29145073/target-induced-proximity-ligation-triggers-recombinase-polymerase-amplification-and-transcription-mediated-amplification-to-detect-tumor-derived%C3%A2-exosomes-in-nasopharyngeal-carcinoma-with-high-sensitivity
#5
Wanli Liu, Jianpei Li, Yixian Wu, Shan Xing, Yanzhen Lai, Ge Zhang
Tumor-derived exosomes (TEXs) are extracellular vesicles that are continuously released into the blood by tumor cells and carry specific surface markers of the original tumor cells. Substantial evidence has implicated TEXs as attractive diagnostic markers for cancer. However, the detection of TEXs in blood at an early tumor stage is challenging due to their very low concentration. Here, we established a method called PLA-RPA-TMA assay that allows TEXs to be detected with high sensitivity and specificity. Based on two proximity ligation assay (PLA) probes that recognize a biomarker on a TEX, we generated a unique surrogate DNA signal for the specific biomarker, which was synchronously amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA), and then the products of the RPA-TMA reaction were quantitatively detected using a gold nanoparticle-based colorimetric assay...
November 9, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/29126443/ubiquitin-c-terminal-hydrolase-isozyme-l1-is-associated-with-shelterin-complex-at-interstitial-telomeric-sites
#6
Aleksandar Ilic, Sumin Lu, Vikram Bhatia, Farhana Begum, Thomas Klonisch, Prasoon Agarwal, Wayne Xu, James R Davie
BACKGROUND: Ubiquitin C-terminal hydrolase isozyme L1 (UCHL1) is primarily expressed in neuronal cells and neuroendocrine cells and has been associated with various diseases, including many cancers. It is a multifunctional protein involved in deubiquitination, ubiquitination and ubiquitin homeostasis, but its specific roles are disputed and still generally undetermined. RESULTS: Herein, we demonstrate that UCHL1 is associated with genomic DNA in certain prostate cancer cell lines, including DU 145 cells derived from a brain metastatic site, and in HEK293T embryonic kidney cells with a neuronal lineage...
November 10, 2017: Epigenetics & Chromatin
https://www.readbyqxmd.com/read/29125601/tm9sf4-is-a-novel-factor-promoting-autophagic-flux-under-amino-acid-starvation
#7
Lei Sun, Zhaoyue Meng, Yifei Zhu, Jun Lu, Zhichao Li, Qiannan Zhao, Yu Huang, Liwen Jiang, Xiaoqiang Yao
Autophagy is a highly complicated process with participation of large numbers of autophagy-related proteins. Under nutrient starvation, autophagy promotes cell survival by breaking down nonessential cellular components for recycling use. However, due to its high complexity, molecular mechanism of autophagy is still not fully understood. In the present study, we report a novel autophagy-related protein TM9SF4, which plays a functional role in the induction phase of autophagic process. TM9SF4 proteins were abundantly expressed in the kidney, especially in renal proximal tubular epithelial cells...
November 10, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/29112683/targeting-de-novo-lipogenesis-as-a-novel-approach-in-anti-cancer-therapy
#8
Katharina Stoiber, Olga Nagło, Carla Pernpeintner, Siwei Zhang, Andreas Koeberle, Melanie Ulrich, Oliver Werz, Rolf Müller, Stefan Zahler, Theobald Lohmüller, Jochen Feldmann, Simone Braig
BACKGROUND: Although altered membrane physiology has been discussed within the context of cancer, targeting membrane characteristics by drugs being an attractive therapeutic strategy has received little attention so far. METHODS: Various acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) inhibitors (like Soraphen A and Cerulenin) as well as genetic knockdown approaches were employed to study the effects of disturbed phospholipid composition on membrane properties and its functional impact on cancer progression...
November 7, 2017: British Journal of Cancer
https://www.readbyqxmd.com/read/29107693/wdr74-participates-in-an-early-cleavage-of-the-pre-rrna-processing-pathway-in-cooperation-with-the-nucleolar-aaa-atpase-nvl2
#9
Nobuhiro Hiraishi, Yo-Ichi Ishida, Haruka Sudo, Masami Nagahama
WD repeat-containing protein 74 (WDR74), a nucleolar-localized protein, is the mammalian ortholog of Nsa1, a 60S ribosome assembly factor in yeast. We previously showed that WDR74 associates with MTR4, the nuclear exosome-assisting RNA helicase, whose dissociation is prohibited by an ATPase-deficient mutant of the AAA-type chaperone NVL2. However, the functions and regulation of WDR74 during ribosome biogenesis in cooperation with NVL2 remains unknown. Here, we demonstrated that knockdown of WDR74 leads to significant defects in the pre-rRNA cleavage within the internal transcribed spacer 1 (ITS1), occurring in an early stage of the processing pathway...
October 28, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29101179/reduced-membrane-cholesterol-following-chronic-hypoxia-limits-orai1-mediated-pulmonary-endothelial-ca-2-entry
#10
Bojun Zhang, Jay S Naik, Nikki L Jernigan, Benjimen R Walker, Thomas C Resta
Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca(2+) entry (SOCE) and diminished Ca(2+)-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAEC) is tightly regulated by membrane cholesterol, and that decreased membrane cholesterol is responsible for impaired SOCE following CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAEC through the interaction of Orai1 and stromal interaction molecule 1 (STIM1)...
November 3, 2017: American Journal of Physiology. Heart and Circulatory Physiology
https://www.readbyqxmd.com/read/29101055/detection-of-post-translational-modifications-using-solid-phase-proximity-ligation-assay
#11
Felipe Marques Souza de Oliveira, Stefan Mereiter, Peter Lönn, Benjamin Siart, Qiujin Shen, Johan Heldin, Doroteya Raykova, Niclas Karlsson, Karol Polom, Franco Roviello, Celso A Reis, Masood Kamali-Moghaddam
Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs...
October 31, 2017: New Biotechnology
https://www.readbyqxmd.com/read/29094393/quantitative-image-analysis-of%C3%A2-protein-expression-and-colocalization-in-skin-sections
#12
Hanqian Zhang, Maja Ericsson, Marie Virtanen, Simone Weström, Carolina Wählby, Anders Vahlquist, Hans Törmä
Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalization analysis, respectively. However, an efficient quantitative method to analyze both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalization in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined...
November 1, 2017: Experimental Dermatology
https://www.readbyqxmd.com/read/29079505/a-constitutively-active-ikk-complex-at-the-axon-initial-segment
#13
Hans-Georg König, Orla Watters, Sinéad Kinsella, Mohammed Ameen, Beau J Fenner, Jochen H M Prehn
BACKGROUND: Previous studies provided evidence for an accumulation of IκB-kinase (IKK) α/β at the axon initial segment (AIS), a neuronal compartment defined by ankyrin-G expression. Here we explored whether the presence of the IKK-complex at the AIS was associated with the activation of IKK signaling at this site. METHODS AND RESULTS: Proximity-ligation assays (PLAs) using pan-IKK α/β, phospho-IKK α/β -specific as well as ankyrin-G specific antibodies validated their binding to proximal epitopes in the AIS, while antibodies to other phosphorylated signaling proteins showed no preference for the AIS...
October 24, 2017: Brain Research
https://www.readbyqxmd.com/read/29070491/rhoa-increases-asic1a-plasma-membrane-localization-and-calcium-influx-in-pulmonary-arterial-smooth-muscle-cells-following-chronic-hypoxia
#14
Lindsay M Herbert, Thomas C Resta, Nikki L Jernigan
Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca(2+) levels and enhanced RhoA/Rho kinase-dependent Ca(2+) sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We have previously shown acid sensing ion channel 1a (ASIC1a)-mediated Ca(2+) entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension...
October 25, 2017: American Journal of Physiology. Cell Physiology
https://www.readbyqxmd.com/read/29069773/visualization-and-quantitation-of-epidermal-growth-factor-receptor-homodimerization-and-activation-with-a-proximity-ligation-assay
#15
Keiichi Ota, Taishi Harada, Kohei Otsubo, Akiko Fujii, Yuko Tsuchiya, Kentaro Tanaka, Isamu Okamoto, Yoichi Nakanishi
OBJECTIVES: Activation of the epidermal growth factor receptor (EGFR) results from receptor homodimerization and autophosphorylation and confers sensitivity to tyrosine kinase inhibitors in some tumors. However, the visual detection and quantitation of activated EGFR in the clinical setting has not been established. MATERIALS AND METHODS: A proximity ligation assay (PLA) was applied to detect EGFR homodimers in non-small cell lung cancer (NSCLC) cell lines and tissue specimens...
September 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/29066953/disturbances-in-the-fgfr1-5-ht1a-heteroreceptor-complexes-in-the-raphe-hippocampal-5-ht-system-develop-in-a-genetic-rat-model-of-depression
#16
Dasiel O Borroto-Escuela, Caitlin M DuPont, Xiang Li, David Savelli, Davide Lattanzi, Ipsit Srivastava, Manuel Narváez, Michael Di Palma, Elisa Barbieri, Yuniesky Andrade-Talavera, Riccardo Cuppini, Yuji Odagaki, Miklos Palkovits, Patrizia Ambrogini, Maria Lindskog, Kjell Fuxe
The FGFR1-5-HT1A heteroreceptor complexes are involved in neuroplasticity in the rat hippocampus and in the mesencephalic raphe 5-HT nerve cells. There exists a 5-HT1A protomer enhancement of FGFR1 protomer signaling. Acute and 10 day treatment with intracerebroventricular (i.c.v.) FGF-2 and the 5-HT1A agonist 8-OH-DPAT produced enhanced antidepressant effects in the forced swim test (FST). We studied in the current work the disturbances in the FGFR1-5-HT1A heterocomplexes in a genetic rat model of depression, the Flinders sensitive line (FSL) rats of Sprague-Dawley (SD) origin, by means of neurochemical, neurophysiological and behavioral techniques...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/29061726/angiotensin-ii-type-1-receptor-mechanoactivation-involves-rgs5-regulator-of-g-protein-signaling-5-in-skeletal-muscle-arteries-impaired-trafficking-of-rgs5-in-hypertension
#17
Kwangseok Hong, Min Li, Zahra Nourian, Gerald A Meininger, Michael A Hill
Studies suggest that arteriolar pressure-induced vasoconstriction can be initiated by GPCRs (G protein-coupled receptors), including the AT1R (angiotensin II type 1 receptor). This raises the question, are such mechanisms regulated by negative feedback? The present studies examined whether RGS (regulators of G protein signaling) proteins in vascular smooth muscle cells are colocalized with the AT1R when activated by mechanical stress or angiotensin II and whether this modulates AT1R-mediated vasoconstriction...
December 2017: Hypertension
https://www.readbyqxmd.com/read/29054453/disruption-of-dopamine-d1-d2-receptor-complex-is-involved-in-the-function-of-haloperidol-in-cardiac-h9c2-cells
#18
L Lencesova, I Szadvari, P Babula, J Kubickova, B Chovancova, K Lopusna, I Rezuchova, Z Novakova, O Krizanova, M Novakova
AIMS: Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP3 receptors (IP3R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP3R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats...
October 17, 2017: Life Sciences
https://www.readbyqxmd.com/read/29053336/interplay-between-ezh2-and-g9a-regulates-cxcl10-gene-repression-in-idiopathic-pulmonary-fibrosis
#19
William R Coward, Oliver J Brand, Alice Pasini, Gisli Jenkins, Alan J Knox, Linhua Pang
Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodelling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. This study explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases, Enhancer of Zest Homolog 2 (EZH2) and G9a, in CXCL10 repression in IPF. By applying chromatin immunoprecipitation (ChIP), Re-ChIP and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated H3K27me3 was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL) and that EZH2 and G9a physically interacted with each other...
October 20, 2017: American Journal of Respiratory Cell and Molecular Biology
https://www.readbyqxmd.com/read/29046633/activity-dependent-phosphorylation-by-camkii%C3%AE-alters-the-ca-2-affinity-of-the-multi-c2-domain-protein-otoferlin
#20
Sandra Meese, Andreia P Cepeda, Felix Gahlen, Christopher M Adams, Ralf Ficner, Anthony J Ricci, Stefan Heller, Ellen Reisinger, Meike Herget
Otoferlin is essential for fast Ca(2+)-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca(2+)/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells...
2017: Frontiers in Synaptic Neuroscience
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