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Paper-based Invasion Assays for Quantifying Cellular Movement in Three-dimensional Tissue-like Structures 三维组织样结构中定量细胞运动的纸质入侵试验
Current protocols in chemical biology Pub Date : 2017-06-19 DOI: 10.1002/cpch.22
C. Chad Lloyd, Matthew W. Boyce, Matthew R. Lockett
{"title":"Paper-based Invasion Assays for Quantifying Cellular Movement in Three-dimensional Tissue-like Structures","authors":"C. Chad Lloyd,&nbsp;Matthew W. Boyce,&nbsp;Matthew R. Lockett","doi":"10.1002/cpch.22","DOIUrl":"10.1002/cpch.22","url":null,"abstract":"<p>To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor-like environment can be experimentally manipulated and probed. The three-dimensional paper-based invasion assays described here simulate poorly vascularized tissue and allow the invasion of cancerous cells to be visualized and quantified. These cultures are easy to assemble and allow multiple invasion assays to be performed in parallel. By using different materials to control gradients formed across the culture, the chemotactic potential of small molecules can be evaluated in a more representative tissue microenvironment. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 14
Local Generation and Imaging of Hydrogen Peroxide in Living Cells 活细胞中过氧化氢的局部生成和成像
Current protocols in chemical biology Pub Date : 2017-06-19 DOI: 10.1002/cpch.20
Yulia A. Bogdanova, Carsten Schultz, Vsevolod V. Belousov
{"title":"Local Generation and Imaging of Hydrogen Peroxide in Living Cells","authors":"Yulia A. Bogdanova,&nbsp;Carsten Schultz,&nbsp;Vsevolod V. Belousov","doi":"10.1002/cpch.20","DOIUrl":"10.1002/cpch.20","url":null,"abstract":"<p>Described here is a localized H<sub>2</sub>O<sub>2</sub> generation-detection system consisting of a yeast <span>D</span>-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H<sub>2</sub>O<sub>2</sub> dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H<sub>2</sub>O<sub>2</sub> generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.20","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 22
Measuring Cancer Drug Sensitivity and Resistance in Cultured Cells 肿瘤培养细胞的药物敏感性和耐药性测定
Current protocols in chemical biology Pub Date : 2017-06-19 DOI: 10.1002/cpch.21
Mario Niepel, Marc Hafner, Mirra Chung, Peter K. Sorger
{"title":"Measuring Cancer Drug Sensitivity and Resistance in Cultured Cells","authors":"Mario Niepel,&nbsp;Marc Hafner,&nbsp;Mirra Chung,&nbsp;Peter K. Sorger","doi":"10.1002/cpch.21","DOIUrl":"10.1002/cpch.21","url":null,"abstract":"<p>Measuring the potencies of small-molecule drugs in cell lines is a critical aspect of preclinical pharmacology. Such experiments are also prototypical of high-throughput experiments in multi-well plates. The procedure is simple in principle, but many unrecognized factors can affect the results, potentially making data unreliable. The procedures for measuring drug response described here were developed by the NIH LINCS program to improve reproducibility. Key features include maximizing uniform cell growth during the assay period, accounting for the effects of cell density on response, and correcting sensitivity measures for differences in proliferation rates. Two related protocols are described: one involves an endpoint measure well-suited to large-scale studies and the second is a time-dependent measurement that reveals changes in response over time. The methods can be adapted to other types of plate-based experiments. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.21","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 32
Photoactivated In Vivo Proximity Labeling 光激活的体内接近标记
Current protocols in chemical biology Pub Date : 2017-06-19 DOI: 10.1002/cpch.18
David B. Beck, Roberto Bonasio
{"title":"Photoactivated In Vivo Proximity Labeling","authors":"David B. Beck,&nbsp;Roberto Bonasio","doi":"10.1002/cpch.18","DOIUrl":"10.1002/cpch.18","url":null,"abstract":"<p>Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein-protein and protein-RNA interactions using IPL. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.18","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Designing Drug-Response Experiments and Quantifying their Results 设计药物反应实验并量化实验结果
Current protocols in chemical biology Pub Date : 2017-06-19 DOI: 10.1002/cpch.19
Marc Hafner, Mario Niepel, Kartik Subramanian, Peter K. Sorger
{"title":"Designing Drug-Response Experiments and Quantifying their Results","authors":"Marc Hafner,&nbsp;Mario Niepel,&nbsp;Kartik Subramanian,&nbsp;Peter K. Sorger","doi":"10.1002/cpch.19","DOIUrl":"10.1002/cpch.19","url":null,"abstract":"<p>We developed a Python package to help in performing drug-response experiments at medium and high throughput and evaluating sensitivity metrics from the resulting data. In this article, we describe the steps involved in (1) generating files necessary for treating cells with the HP D300 drug dispenser, by pin transfer or by manual pipetting; (2) merging the data generated by high-throughput slide scanners, such as the Perkin Elmer Operetta, with treatment annotations; and (3) analyzing the results to obtain data normalized to untreated controls and sensitivity metrics such as IC<sub>50</sub> or GR<sub>50</sub>. These modules are available on GitHub and provide an automated pipeline for the design and analysis of high-throughput drug response experiments, that helps to prevent errors that can arise from manually processing large data files. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.19","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35099906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 21
UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis UbFluor:一种监测HECT E3连接酶催化的荧光硫酯
Current protocols in chemical biology Pub Date : 2017-03-02 DOI: 10.1002/cpch.17
David T. Krist, Peter K. Foote, Alexander V. Statsyuk
{"title":"UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis","authors":"David T. Krist,&nbsp;Peter K. Foote,&nbsp;Alexander V. Statsyuk","doi":"10.1002/cpch.17","DOIUrl":"10.1002/cpch.17","url":null,"abstract":"<p>HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3∼Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.17","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39983095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
A General Non-Radioactive ATPase Assay for Chromatin Remodeling Complexes 一般非放射性三磷酸腺苷酶测定染色质重塑复合体
Current protocols in chemical biology Pub Date : 2017-03-02 DOI: 10.1002/cpch.16
Benjamin Z. Stanton, Courtney Hodges, Gerald R. Crabtree, Keji Zhao
{"title":"A General Non-Radioactive ATPase Assay for Chromatin Remodeling Complexes","authors":"Benjamin Z. Stanton,&nbsp;Courtney Hodges,&nbsp;Gerald R. Crabtree,&nbsp;Keji Zhao","doi":"10.1002/cpch.16","DOIUrl":"10.1002/cpch.16","url":null,"abstract":"<p>Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF-like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function. The protocol in this article defines the first highly quantitative ATPase assay for intact remodeling complexes that does not require radiation or reconstitution of recombinantly expressed subunits. This protocol is specifically useful for defining the catalytic role of active-site mutations in the context of other regulatory subunits and quantitatively rank-ordering inactivating catalytic-site mutations. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.16","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39983560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay 利用哺乳动物膜双杂交(MaMTH)试验检测膜蛋白-蛋白相互作用
Current protocols in chemical biology Pub Date : 2017-03-02 DOI: 10.1002/cpch.15
Punit Saraon, Ingrid Grozavu, Sang Hyun Lim, Jamie Snider, Zhong Yao, Igor Stagljar
{"title":"Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay","authors":"Punit Saraon,&nbsp;Ingrid Grozavu,&nbsp;Sang Hyun Lim,&nbsp;Jamie Snider,&nbsp;Zhong Yao,&nbsp;Igor Stagljar","doi":"10.1002/cpch.15","DOIUrl":"10.1002/cpch.15","url":null,"abstract":"<p>Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes. In the protocols below, we describe a newly developed membrane protein interaction assay called the Mammalian-Membrane Two-Hybrid (MaMTH), designed specifically for the detection of integral membrane PPIs in the context of living mammalian cells. Prior to using MaMTH, cell lines of interest are genetically modified to encode a reporter of choice. MaMTH “bait” and “prey” constructs of interest are also generated using Gateway cloning technology. The assay is then performed by co-transfection of baits and preys, with bait-prey interaction quantifiably assessed by way of a reporter signal (e.g., light (luciferase), fluorescence (GFP). © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39982884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 16
Enriching s4U-RNA Using Methane Thiosulfonate (MTS) Chemistry 甲烷硫代磺酸盐(MTS)化学富集s4U-RNA
Current protocols in chemical biology Pub Date : 2016-12-07 DOI: 10.1002/cpch.12
Erin E. Duffy, Matthew D. Simon
{"title":"Enriching s4U-RNA Using Methane Thiosulfonate (MTS) Chemistry","authors":"Erin E. Duffy,&nbsp;Matthew D. Simon","doi":"10.1002/cpch.12","DOIUrl":"10.1002/cpch.12","url":null,"abstract":"<p>Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s<sup>4</sup>U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s<sup>4</sup>U-RNA is the efficiency of the chemistry used to enrich s<sup>4</sup>U-RNA. Here, we present a protocol for s<sup>4</sup>U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s<sup>4</sup>U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s<sup>4</sup>U to study RNA biology. © 2016 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"8 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.12","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50803085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 30
Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin 鸟嘌呤核苷酸交换因子(GIV/Girdin)的生物化学、生物物理和细胞技术研究
Current protocols in chemical biology Pub Date : 2016-12-07 DOI: 10.1002/cpch.13
Pradipta Ghosh, Nicolas Aznar, Lee Swanson, I-Chung Lo, Inmaculada Lopez-Sanchez, Jason Ear, Cristina Rohena, Nicholas Kalogriopoulos, Linda Joosen, Ying Dunkel, Nina Sun, Peter Nguyen, Deepali Bhandari
{"title":"Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin","authors":"Pradipta Ghosh,&nbsp;Nicolas Aznar,&nbsp;Lee Swanson,&nbsp;I-Chung Lo,&nbsp;Inmaculada Lopez-Sanchez,&nbsp;Jason Ear,&nbsp;Cristina Rohena,&nbsp;Nicholas Kalogriopoulos,&nbsp;Linda Joosen,&nbsp;Ying Dunkel,&nbsp;Nina Sun,&nbsp;Peter Nguyen,&nbsp;Deepali Bhandari","doi":"10.1002/cpch.13","DOIUrl":"10.1002/cpch.13","url":null,"abstract":"<p>Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about noncanonical G protein activation by GIV and its relevance in health and disease. © 2016 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":38051,"journal":{"name":"Current protocols in chemical biology","volume":"8 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2016-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpch.13","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50803136","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
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