Estrogen Receptors: Methods and Protocols (Methods in Molecular Biology, 2418)

Description

This fully updated collection includes chapters on a wide array of techniques that are vital for advancing our understanding of the physiological and pathological effects of estrogen, mediated by estrogen receptor α (ERα) and estrogen receptor β (ERβ) as well as the non-genomic membrane-bound G-protein coupled estrogen receptor (GPER/GPR30). The protocols range from standard methods and important laboratory workhorses such as immunoblot and RT-qPCR, to newer technologies such as PET imaging, reporter animal models, polysome fractionation, and many others, involving a broad range of tissue types to demonstrate the variety of estrogen receptor effects. Written for this highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Estrogen Receptors: Methods and Protocols, Second Edition serves as an ideal guide to researchers working to explore the vibrancy and excitement of the field of estrogen receptors and estrogen actions. Preface Contents Contributors Chapter 1: Antibody Validation for Estrogen Receptor Beta 1 Introduction 2 Materials 2.1 Antibodies 2.2 Tissue Sections and Cells 2.3 Immunohistochemistry 2.4 Western Blot (WB) 2.5 Immunoprecipitation (IP) for Mass Spectrometry (MS) Analysis (Optional) 3 Methods 3.1 Cell and Tissue Sectioning 3.2 Immunohistochemistry and Annotation 3.3 Western Blot 3.4 Immunoprecipitation Followed by Mass Spectrometry 4 Notes References Chapter 2: Immunoblot Detection of the Phosphorylation of the Estrogen Receptor α as an Outcome of GPR30/GPER1 Activation 1 Introduction 2 Materials 2.1 Components for Creating Lysates 2.2 SDS Polyacrylamide Gel Components 2.3 Immunoblotting Components 2.4 Antibodies and Stripping Buffer 2.5 Hormone Treatments and Animal Work 3 Methods 3.1 Tissue Procurement 3.2 Making the Lysate 3.3 Making Two Gels 3.4 Gel Electrophoresis and Blotting 3.5 Blocking and Antibody Incubation 4 Notes References Chapter 3: Immunoprecipitation Analyses of Estrogen Receptor α Phosphorylated at Serine 216 in the Mouse Liver 1 Introduction 2 Materials 3 Methods 3.1 Animals 3.2 Chromatin Immunoprecipitation Assay (ChIP) 3.3 Cell Culture and Transfection 3.4 Co-immunoprecipitation (Co-IP) and Western Blot Analyses 4 Notes References Chapter 4: Immunofluorescent Verification of Silencing Estrogen Receptor α with siRNA in the Intact Rodent Brain 1 Introduction 2 Materials 2.1 Animals 2.2 Viral Vectors Containing shRNA for ERα and Luciferase 2.3 Stereotaxic and Microinfusion Materials 2.4 Immunocytochemical Verification of shRNA Suppression 2.5 Quantitative Imaging 3 Methods 3.1 Delivery of Viral Vector shRNA to Animals 3.2 Immunofluorescent Verification of Silencing Efficiency 4 Notes References Chapter 5: Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils 1 Introduction 2 Materials 3 Methods 3.1 In Vitro Phosphorylation ERα Proteins by PKC 3.2 Immunohistochemistry of Uterine Sections 3.3 Competitive Immunohistochemistry of Uterine Sections 3.4 Preparation of Peripheral White Blood Cell (WBC) Fractions 3.5 Double Fluorescence Staining with Fluorescein and Texas Red 3.6 Double Fluorescence Staining with Alexa 488 and Alexa 594 3.7 In Vitro Migration Assay 4 Notes References Chapter 6: Quantification of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate in Response to Estrogen 1 Introduction 2 Materials 2.1 Buffers and Blocking Reagents for Standard Immunohistochemistry 2.2 Reagents for Standard Immunohistochemistry 2.3 Equipment for Standard Immunohistochemistry and PLA 2.4 Antibodies and Detection Reagents for Standard Immunohistochemistry and PLA 3 Methods (See Note 3) 3.1 Deparaffinization (See Note 5) 3.2 Antigen Retrieval (See Note 6) 3.3 Blocking (See Note 3) 3.4 Primary Antibody Incubation (See Note 3) 3.5 DAB Alternative Detection (Secondary Antibody Incubation, See Note 8) 3.6 PLA Probes (Secondary Antibody Incubation) 3.7 Ligation of Oligonucleotide Conjugates 3.8 Rolling Circle Amplification and Detection (See Note 14) 3.9 Final Wash (See Note 15) 4 Notes References Chapter 7: In Situ Hybridization of Estrogen Receptors α and β in the Human Testis 1 Introduction 2 Materials 2.1 Media 2.2 Stock Solutions 2.3 Working Solutions for In Situ Hybridization 2.4 cRNA Probe Preparation 2.5 In Situ Hybridization 2.6 Other Reagents and Equipment 3 Methods 3.1 Preparation of PCR Product for RNA Probe Synthesis 3.2 Ligation of PCR Product into the Vector and Transformation of Competent Cells 3.3 Plasmid Purification and Digestion 3.4 In Vitro Transcription 3.5 Preparation of Histological Sections 3.6 In Situ Hybridization: Preparation of Sections and Hybridization Reaction 3.7 In Situ Hybridization: Washing and Immunohistochemistry 3.8 In Situ Hybridization: Signal Detection 4 Notes References Chapter 8: Extraction of RNA and Analysis of Estrogen-Responsive Genes by RT-qPCR 1 Introduction 2 Materials 2.1 Materials for RNA Extraction and RNA Hygiene 2.2 Materials for Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) 3 Methods 3.1 Tissue Procurement 3.2 RNA Hygiene and RNA Extraction 3.3 Analysis and Quantitation of RNA 3.4 One-Step RT-qPCR 4 Notes References Chapter 9: PET Imaging of Estrogen Receptors Using 18F-Based Radioligands 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment and Supplies 3 Methods 4 Notes References Chapter 10: The Use of ERE-Luc Reporter Mice to Monitor Estrogen Receptor Transcriptional Activity in a Spatio-Temporal Dimens... 1 Introduction 2 Materials 2.1 Mice and Diet 2.2 Mouse Treatments 2.3 In Vivo and Ex Vivo Bioluminescence Imaging 2.4 Luciferase Enzymatic Assay 2.5 General Items 2.6 Analysis of Imaging Data and Statistical Analysis 3 Methods 3.1 In Vivo Imaging Setup: Luciferase Substrate Distribution 3.1.1 Time-Course Study (See Note 4) 3.1.2 Dose-Response Study (See Note 4) 3.2 In Vivo Imaging Setup: Dose-Response Analysis 3.3 In Vivo Imaging Setup: Identification of the Sample Size by Power Analysis 3.4 In Vivo Imaging: Spatio-Temporal Analysis of Long-Term Exposure to SERMs 3.5 Ex Vivo Assessment of Luciferase Enzymatic Activity in Brain Areas 3.6 Ex Vivo Quantitative Assessment of Luciferase Enzymatic Activity in Tissue Extracts 3.7 How to Analyze the Potency of a Given SERM by Clustering Analysis 4 Notes References Chapter 11: Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model 1 Introduction 2 Materials 2.1 Solutions for Zebrafish Embryo Work 2.2 Chemicals 3 Methods 3.1 Zebrafish Maintenance 3.2 Breeding of Fish and Collection of Fertilized Eggs 3.3 Embryo Treatments 3.4 Imaging and Quantification of Tissue-Specific Fluorescence 3.5 Fluorescence Measurements 3.6 Measuring Estrogen Receptor Antagonism 4 Notes References Chapter 12: Assessing Estrogenic Activity of Classical Estrogen Receptor-Binding Compounds 1 Introduction 2 Materials 3 Methods 3.1 Estrogen Response Element (ERE) Transcriptional Activity 3.2 MCF-7 Cell Proliferation and Viability 3.3 ERα Degradation 4 Notes References Chapter 13: An Optimized ChIP-Seq Protocol to Determine Chromatin Binding of Estrogen Receptor Beta 1 Introduction 2 Materials 2.1 Cell Line Model 2.2 Cell Culture Media and Treatment 2.3 Buffers for ChIP 2.4 Plastic Ware 2.5 Equipment 2.6 Reagents 2.7 Preparation of Reagents 3 Methods 3.1 Cell Culture, Serum Deprivation, and Ligand Treatment 3.2 Crosslinking Cells (Day 1) 3.3 Prepare Dynabeads for ChIP (Day 1) 3.4 Cell Lysis (Day 1) 3.5 Chromatin Shearing (Day 1) 3.6 Pre-Block and Incubation with Antibody (Day 1) 3.7 ChIP (Day 2) 3.8 DNA Purification (Day 3) 3.9 ChIP-Seq Data Analysis 4 Notes References Chapter 14: Polysome Fractionation for Transcriptome-Wide Studies of mRNA Translation 1 Introduction 1.1 Transcriptome-Wide Studies of mRNA Translation Reveal Selective Modulation of Protein Synthesis Downstream of ERα 1.2 Insights into mRNA Translation Are Obscured if Proteomes Are Compared to Transcriptomes 1.3 Transcriptome-Wide Quantification of mRNA Translation 1.4 Polysome-Profiling: An Overview 2 Materials 2.1 Reagents 2.2 Instruments 2.3 Equipment Needed for Working with Frozen Tissue 3 Methods 3.1 Experimental Design for Polysome Profiling 3.2 Preparation of Sucrose Gradients 3.2.1 Preparation of a Linear Sucrose Gradient 3.2.2 Preparation of an Optimized Sucrose Gradient 3.3 Isolating Cytosolic Extracts from Cell Cultures and Loading these on Sucrose Gradients 3.3.1 Setting up Cell Culture Conditions 3.3.2 Cell Harvest, Cell Lysis, and Loading Lysates on Sucrose Gradients 3.4 Isolating Cytosolic Extracts from Frozen Tissues and Loading these on Sucrose Gradients 3.5 Ultracentrifugation 3.6 Fractionating Sucrose Gradients 3.7 RNA Extraction from Sucrose Gradients 3.8 RNA Extraction from Fractionated Samples with Low RNA Amounts 3.9 Quantification of Gene Expression 4 Notes References Chapter 15: Anota2seq Analysis for Transcriptome-Wide Studies of mRNA Translation 1 Introduction 1.1 Identifying Changes in Translation Efficiency on a Transcriptome-Wide Scale 1.2 Analysis of Translation Activity Using Anota2seq 2 Setting up R and Performing Basic Data Quality Control 2.1 Installing R, Rstudio, Bioconductor, and Anota2seq 2.1.1 Install R and Rstudio 2.1.2 Install Bioconductor and Anota2seq 2.2 Data Import and Formatting 2.2.1 Setting your Working Directory and Importing Data 2.2.2 Formatting Data for Anota2seq Analysis 2.3 Basic Quality Control of Sample Classes 3 Running an Anota2seq Analysis 3.1 Anota2seq Overview 3.2 Generate an Anota2seq Data Set Object 3.3 Performing Anota2seq Quality Control to Test Model Assumptions 3.4 Performing the Analysis of Differences in Translation Efficiency 3.5 Plot p-Value and Adjusted p-Value Densities to Get an Overview of how Gene Expression Is Regulated at Different Levels 3.6 Apply Significance Thresholds to Identify Regulated mRNAs 3.7 Determine Modes of Gene Expression Regulation 3.8 Visualize Gene Expression Modes in a Fold Change Plot 3.9 Extract Output Tables from the Anota2seq Object 3.10 Analysis beyond Anota2seq 3.10.1 Investigate 5′ UTR Features 3.10.2 Overrepresentation or Gene Set Enrichment Analysis 4 Notes References Chapter 16: Whole-Genome Genotyping Using DNA Microarrays for Population Genetics 1 Introduction 2 Materials 2.1 DNA Extraction and Quantitation 2.2 DNA Prep and Microarray Workflow 2.3 Data Generation, QC, and Analysis 3 Methods 3.1 DNA Extraction and Quantitation 3.2 DNA Prep and Microarray Workflow 3.3 Data QC and Analysis 4 Notes References Chapter 17: Proteomics Analysis of the Estrogen Effects in the Rat Uterus Using Gel-LC and Tandem Mass Spectrometry Approaches 1 Introduction 2 Materials 3 Methods 3.1 Sample Preparation 3.2 Gel Staining Procedure 3.3 Gel Imaging and Documentation 3.4 In Gel Digestion 3.5 Peptide Analysis by 1D-NanoUHPLC and Tandem Mass Spectrometry 3.6 Bioinformatics Analysis of Data 4 Notes References Chapter 18: Expression Profiles of Estrogen-Regulated MicroRNAs in Cancer Cells 1 Introduction 2 Materials 2.1 Cell Line Model 2.2 Cell Culture Media and Treatment 2.3 RNA Extraction, cDNA Synthesis, and qPCR for mRNA Reagents 2.4 miRNA Microarray 2.5 Low-Density Array qPCR Analysis 2.6 Small RNA-Seq Analysis 2.7 SYBR/EBA Green Confirmatory miRNA qPCR 2.8 TaqMan Confirmatory miRNA qPCR 3 Methods 3.1 Cell Culture and Treatment 3.2 RNA Extraction and Quality Control 3.3 miRNA Profiling Analysis: miRNA Microarray (See Fig. 2a) 3.4 Low-Density Array qPCR 3.5 miRNA-Seq 3.6 SYBR/EBA Green Confirmatory qPCR Analysis of Regulated miRNAs 3.7 TaqMan qPCR Confirmatory Analysis of Regulated miRNAs 4 Notes References Chapter 19: Molecular Cloning and Purification of the Protein Lysine Methyltransferase SMYD2 and its Co-crystallization with a... 1 Introduction 2 Materials 2.1 Stock Solutions 2.2 Molecular Cloning 2.3 Protein Expression 2.4 Protein Purification 2.5 Crystallization 3 Methods 3.1 Molecular Cloning 3.2 Large-Scale Protein Expression (See Note 12) 3.3 Harvest Cells 3.4 Lyse Cells 3.5 Protein Purification 3.6 Crystallization 4 Notes References Chapter 20: Molecular Design, Synthesis, and Evaluation of SNIPER(ER) that Induces Targeted Protein Degradation of ERα 1 Introduction 1.1 Design and Synthesis of SNIPER(ER) 1.2 ERα Protein Degradation 1.3 Growth Inhibition and Cell Death 1.4 Antitumor Activity in Xenograft Model 2 Materials 2.1 Design and Synthesis of SNIPER(ER) 2.2 ERα Protein Degradation 2.3 Growth Inhibition and Cell Death 2.4 Antitumor Activity in Xenograft Model 3 Methods 3.1 Design and Synthesis of SNIPER(ER)-1 (8), -2 (9), and -3 (10) 3.2 Design and Synthesis of SNIPER(ER)-19 (14) 3.3 Design and Synthesis of SNIPER(ER)-105 (17) 3.4 Design and Synthesis of SNIPER(ER)-87 (21), -110 (28), -113 (29), -119 (30), and -126 (33) 3.5 ERα Protein Degradation 3.6 Growth Inhibition and Cell Death 3.7 Antitumor Activity in Xenograft Model 4 Notes References Chapter 21: Assessment of Metabolic Regulation by Estrogen Receptors 1 Introduction 2 Materials 2.1 Isolation of Adipocytes and Stromal Vascular Fraction 2.2 Differentiation of Preadipocytes and Quantification of Lipids 2.3 Glucose Uptake 2.4 Glucose and Insulin Testing 2.5 Islet Isolation 2.6 Agilent Seahorse Mitochondrial Stress Test 3 Methods 3.1 Adipocyte and Stromal Vascular Fraction Isolation from Adipose Tissue 3.2 Differentiation of Preadipocytes (3T3-L1 or Primary Cells) 3.3 Glucose Uptake in Adipocytes/Adipose Tissue 3.4 Intraperitoneal Glucose (IPGTT) and Insulin Tolerance Test (IPITT) 3.5 Fasting Plasma Glucose and Insulin Levels and Calculation of Insulin Resistance Index 3.6 Islet Isolation 3.7 Real-Time Live Cell Mitochondrial Function Using Seahorse 4 Notes References Chapter 22: RNA-Seq Experiment and Data Analysis 1 Introduction 2 Materials 3 Methods 3.1 RNA-Seq Experiment 3.2 RNA-Seq Data Analysis 4 Notes References Index