HDAC/HAT Function Assessment and Inhibitor Development: Methods and Protocols

Description

This fully updated edition provides a series of methods for how best to assess functions of histone deacetylases and acetyltransferases. The disease-relevance of dysregulated protein deacetylation by overexpressed or aberrantly activated histone deacetylases has spurred an intense search for novel and improved inhibitors of these enzymes, as reflected in this collection. Expert contributors explore the generation and evaluation of novel histone deacetylase inhibitors and new and improved techniques to assess acetylation-dependent molecular mechanisms in vitro and in vivo. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and up-to-date, HDAC/HAT Function Assessment and Inhibitor Development: Methods and Protocols, Second Edition serves as an ideal guide for researchers seeking to further elucidate this vital area of study. Preface Contents Contributors Part I: In Vivo and 3R Models to Assess HDAC Functions and Effects of HDACi Chapter 1: Analyzing Lymphoma Development and Progression Using HDACi in Mouse Models 1 Introduction 2 Materials 2.1 Inhibitors and Solutions 2.2 Mice and Injection 2.3 Assessment of Pharmacological Tolerance 2.4 Tumor Excision and Cell Isolation 2.5 Flow Cytometry 3 Methods 3.1 Preparation of Inhibitors 3.2 Injection of Mice 3.3 Analysis of Pharmacological Tolerance 3.4 Macroscopic Analysis of Tumor Formation 3.5 Analysis of Lymphopoiesis and Tumor Phenotype 4 Notes References Chapter 2: Colony Formation Assay to Test the Impact of HDACi on Leukemic Cells 1 Introduction 2 Materials 2.1 Reagent Preparation 2.2 Sample Preparation 2.3 Quantification 3 Methods 3.1 Reagent Preparation 3.2 Sample Preparation 4 Notes References Untitled Chapter 3: Defined Human Leukemic CD34+ Liquid Cultures to Study HDAC/Transcriptional Repressor Complexes 1 Introduction 2 Materials 2.1 Culture of Human Bone Marrow CD34+ Progenitor Cells 2.2 Produce Retroviral Particles for Oncogene Expression (Transfection) 2.3 Retroviral Transduction of Human CD34+ Progenitor Cells 2.4 Characterize the Transduced Progenitor Cells: Monitoring Outgrowth 2.5 Characterize the Transduced Progenitor Cells: Immunophenotyping, Morphology and Colony Forming Units (CFUs) 3 Methods 3.1 Culture of Human Bone Marrow CD34+ Progenitor Cells 3.2 Production of Retroviral Particles for Oncogene Expression 3.3 Retroviral Transduction of CD34+ Human Progenitor Cells 3.4 Characterize the Transduced Progenitor Cells: Monitoring Outgrowth 3.5 Characterize the Transduced Progenitor Cells: Immunophenotyping, Morphology, and Colony-Forming Units (CFUs) 3.5.1 Immunophenotyping 3.5.2 Morphology 3.5.3 Colony-Forming Unit (CFU) Assay 4 Notes References Chapter 4: Development of a Cellular Model Mimicking Specific HDAC Inhibitors 1 Introduction 2 Materials 2.1 Mutagenesis PCR 2.2 Plasmid Preparation 2.3 HAP1 Cell Culturing 2.4 Stable Transfection and Selection of the Targeted Clones 2.5 Validation of Targeted Clones 2.5.1 Genomic DNA Extraction 2.5.2 Validation by PCR and Sequencing 2.6 Validation of Protein Expression 3 Methods 3.1 Inserting Mutations into the Open Reading Frames of HDACs 3.2 Plasmid Preparation 3.3 Recommendations for Plasmid Design/Preparation 3.4 HAP1 Cell Culture and Preparation for Genetic Targeting 3.5 Targeting of HAP1 Cells 3.5.1 Targeted Gene Disruption and the Generation of Class I HDAC KO Cells (Fig. 5a) 3.5.2 Insertion of Transgenic Class I HDACs by Homologous Recombination into a Safe Harbor Locus (Fig. 5b) 3.5.3 Preparation of Transfection Reagents 3.5.4 Transfection of HAP1 Cells 3.6 Selection of Correctly Targeted Clones 3.7 Limiting Dilution 3.8 Clone Picking and Expansion 3.9 Clone Freezing 3.10 Validation of Targeted Gene Disruption/Insertion on DNA Level 3.10.1 Genomic DNA Extraction to Verify the Targeted Cell Lines 3.10.2 PCR Amplification and Sanger Sequencing 3.11 Validation of Targeted Gene Disruption/Insertion on mRNA Level 3.12 Validation of Targeted Gene Disruption/Insertion on Protein Level 3.12.1 Western Blotting Analysis 3.12.2 Immunofluorescence Analysis 3.13 Stabilization of HAP1 Genetic Background 4 Notes References Chapter 5: Evaluation of Antitumor and On-Target Activity of HDAC Inhibitors with the Zebrafish Embryo Xenograft Model 1 Introduction 2 Materials 2.1 General Supplies 2.2 Xenotransplantation 2.3 Imaging and Toxicity Assay 2.4 Immunohistochemistry 3 Methods 3.1 Zebrafish Handling 3.2 Toxicity Assays Prior Xenotransplantation 3.3 Cell Preparation and Zebrafish Xenotransplantation 3.4 Treatment 3.5 Imaging and Analysis 3.6 Immunohistochemistry (IHC) 4 Notes References Chapter 6: Human Platelet Lysate as Valid Cell Growth Additive to Assess Protein Acetylation 1 Introduction 2 Materials 3 Methods 3.1 Production of hPL 3.2 Adaptation of the Cells 3.3 Freezing of Adapted Cells 3.4 Assessment of Acetylation in Cells That Grow in hPL-Supplemented Medium 4 Notes References Chapter 7: Investigating Physiopathological Roles for Sirtuins in a Mouse Model 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 3 Methods 3.1 Sirt7-/- Allele Generation 3.2 Genotyping 3.3 Preparation of Mouse Embryonic Fibroblasts (MEFs) 3.4 Replicative Lifespan Assay 3.5 Senescence-Associated β-Galactosidase (SA-β-gal) Staining 3.6 Immunofluorescence Staining 3.7 Hematoxylin-Eosin (H&E) Staining 3.8 Masson Trichrome Staining 3.9 Bone Density Determination 3.10 Myography 3.11 Neovasculogenesis 3.12 Glucose Tolerance Test 3.13 Morris Water Maze 3.14 Transthoracic Echocardiography 3.15 Endurance Running Test 3.16 Statistical Analyses 4 Notes References Chapter 8: Patient-Derived Organoids for In Vivo Validation of In Vitro Data 1 Introduction 2 Materials 2.1 Equipment 2.2 Proliferation and Differentiation Medium for CRC-Originating PDOs 2.3 Washing Buffer for CRC-Originating PDOs 3 Methods 3.1 Tissue Processing 3.2 Organoid Freezing 3.3 Organoid Splitting 3.4 Cell Recovery 3.5 PDO Implantation Into Mice 3.6 Monitoring Tumor Outgrowth 3.7 Explantation 3.8 Tumor Preparation and Conservation 4 Notes References Part II: Novel HDAC Inhibitors and Improved Formulations for HDAC Inhibitors Chapter 9: Analysis of HDACi-Coupled Nanoparticles: Opportunities and Challenges 1 Introduction 2 In Vitro Analyses and Biological Characterization 2.1 Cellular Treatment 2.2 Imaging and Analysis of Nanoparticle Uptake and Cargo Distribution 2.3 Investigation of the In Vitro and Ex Ovo Biocompatibility 2.4 Investigation of Drug Release and Biological Activity 3 Outlook References Chapter 10: Development of Pyrazine-Anilinobenzamides as Histone Deacetylase HDAC1-3 Selective Inhibitors and Biological Testi... 1 Introduction 2 Materials 2.1 Component for Alkylation 2.2 Components for Reductive Amination 2.3 Components for Synthesis of Different Carboxylic Acids Through Ester Hydrolysis 2.4 Components for Amide Coupling 2.5 Components for Cleavage of Boc Group 2.6 Chromatography Equipment 2.7 Enzymatic Testing of HDAC Inhibitors 2.8 Cell Culture 2.9 Viability Assay 3 Methods 3.1 Alkylation 3.2 Reductive Amination 3.3 Synthesis of Different Carboxylic Acids Through Ester Hydrolysis 3.4 Preparation of Aminobenzamides by Amide Coupling of the Appropriate Carboxylic Acids and Corresponding Amines, Followed by... 3.5 Enzymatic Testing of HDAC Inhibitors 3.6 Biological Assessment with a Proliferation Assay 4 Notes References Chapter 11: Evaluation of Small-Molecule HDAC Inhibitors Through In Vitro and In Cellulo Approaches 1 Introduction 2 Materials 2.1 PAMPA Assay 2.1.1 Reagents and Solutions 2.1.2 Equipment 2.1.3 Assay Plates 2.2 Cytotoxicity Assays 2.2.1 Materials 2.2.2 Equipment 2.2.3 Reagents 2.3 Western Blotting 2.3.1 Materials 2.3.2 Equipment 2.3.3 Reagents and Buffers 2.4 Whole Blood Stability 2.4.1 Reagents 2.4.2 Equipment 2.4.3 Assay Plates 3 Methods 3.1 Passive Permeability Evaluation 3.1.1 PAMPA Assay 3.1.2 LC-MS/MS Method 3.1.3 Data Analysis 3.2 Evaluation of Cytotoxicity 3.2.1 Thawing and Culturing Cells 3.2.2 Counting and Seeding Cells 3.2.3 Drug Dosing and Data Collection 3.3 Western Blotting 3.3.1 Counting and Seeding Cells 3.3.2 Drug Dosing 3.3.3 Harvesting Cells 3.3.4 Protein Quantification by BCA (Bicinchoninic Acid) Protein Assay 3.3.5 Gel Electrophoresis (SDS-PAGE) 3.3.6 Transfer the Samples from the Gel to the Membrane and Probe for Specific Biomarkers 3.4 Whole Blood Stability Assay 3.4.1 Blood Stability Sample Preparation 3.4.2 LC-MS/MS Method 3.4.3 Data Analysis 4 Notes References Chapter 12: Synthesis, Biochemical, and Cellular Evaluation of HDAC6 Targeting Proteolysis Targeting Chimeras 1 Introduction 2 Materials 2.1 Acid Chloride Formation 2.2 Preparation of Pomalidomide-Linker Conjugate (6a) 2.3 Preparation of PROTACs with Triazole-Based Linker 2.4 Preparation of Aminoalkanoic Acid Tert-Butyl Ester (4a and 4b) 2.5 Preparation of Pomalidomide-Linker Conjugate (6b and 6c) 2.6 Preparation of PROTACs with Aliphatic Linker 2.7 Chromatography Equipment 2.8 Enzyme Testing 2.9 Cellular Testing 3 Methods 3.1 Acid Chloride Formation 3.2 Preparation of Pomalidomide-Linker Conjugate (6a) 3.3 Preparation of PROTACs with Triazole-Based Linker 3.4 Preparation of Aminoalkanoic Acid Tert-Butyl Ester (4a and 4b) 3.5 Preparation of Pomalidomide-Linker Conjugate (6b and 6c) 3.6 Preparation of PROTACs with Aliphatic Linker 3.7 Enzyme Testing 3.8 Cellular Testing 3.9 Results 4 Notes References Chapter 13: HDACi Delivery Systems Based on Cellulose Valproate Nanoparticles 1 Introduction 2 Materials 2.1 Solvents and Further Substances for Synthesis 2.2 Nuclear Magnetic Resonance (NMR) Analysis 2.3 Scanning Electron Microscopy (SEM) Imaging 2.4 Dynamic Light Scattering (DLS) 2.5 Elemental Analysis (EA) 2.6 Fourier Transform Infrared Spectroscopy (FTIR) 2.7 Size Exclusion Chromatography (SEC) 2.8 Sonifier 3 Methods: Synthesis, Characterization, and Nanoparticle Formation 3.1 Synthesis of Cellulose Valproate (CV) 3.2 Peracetylation of Cellulose Valproate 3.3 Sulfation of Cellulose Valproate (CV-S) 3.4 Synthesis of Cellulose Valproate 6-Aminohexanoate (CV-A) 3.5 Rhodamine B Isothiocyanate Labelling of Cellulose Valproate Sulfate (CV-S-RB) 3.6 Characterization of the Molecular Structure 3.6.1 FTIR Spectroscopy 3.6.2 NMR Spectroscopy 3.6.3 Elemental Analysis 3.7 Characterization of Macromolecular Structure by Size Exclusion Chromatography 3.8 Preparation of Nanoparticles by Emulsification-Evaporation Method 3.9 Characterization of Nanoparticles 3.9.1 Dynamic Light Scattering 3.9.2 Scanning Electron Microscopy 3.10 In Vitro Characterization of Drug Release by Lipase Treatment 4 Notes References Chapter 14: Synthesis and Characterization of Reversible Covalent HDAC4 Inhibitors 1 Introduction 2 Materials 2.1 Chemicals 2.2 Proteins 2.3 Solutions 2.4 Consumables 2.5 Instruments 3 Methods 3.1 Knoevenagel Condensation to Cyanoacrylates 3.2 General Procedure for Ester Saponification 3.3 General Procedure for Amide Formation 3.4 General Procedure for Hydroxamic Acid Formation 3.5 Recombinant Production and Purification of the Catalytic Domain of HDAC4 (cHDAC4) 3.6 HDAC4 Activity Assay 3.7 Spectroscopic Binding and Reversibility Assay 3.8 Jump-Dilution Reversibility Assay 3.9 General Procedure for NMR Reversibility Experiments 4 Notes References Part III: Innovative Techniques to Determine the Impact of Acetylation on Chromatin, DNA Damage, and Cell Stress Responses Chapter 15: Analysis of Cell Cycle and DNA Compaction Dependent Subnuclear Distribution of Histone Marks 1 Introduction 2 Materials 2.1 Cell Culture 2.2 RFi Labeling and Detection 2.3 Immunofluorescence 2.4 Microscopy and Image Analysis 3 Methods 3.1 Cell Culture 3.2 Immunostaining 3.3 Image Acquisition 3.4 Image Preprocessing in FiJi 3.5 Image Analysis in R (Workflow for a Single Cell) 3.6 Image Analysis in R (Workflow on a Batch of Cells) 4 Notes References Chapter 16: Analyzing the Effects of HDAC Inhibitors on DNA Damage and Associated Cytotoxicity in Primary Hepatocytes 1 Introduction 2 Materials 2.1 General Considerations 2.2 Material 2.3 Solutions 3 Methods 3.1 Liver Perfusion 3.1.1 Preparations 3.1.2 Anesthesia 3.1.3 Perfusion In Situ 3.1.4 Isolation of Hepatocytes 3.1.5 Determination of Cell Count and Vitality 3.1.6 Seeding of Hepatocytes 3.2 Alamar Blue Assay 3.3 Immunofluorescence Staining of yH2AX in Primary Hepatocytes and Confocal Laser Scanning Microscopy 4 Notes References Chapter 17: Assessment of HDAC Inhibitor-Induced Endoplasmic Reticulum (ER) Stress 1 Introduction 2 Materials 2.1 Western Blot Analysis of ER Proteins 2.1.1 ER Isolation 2.1.2 Antibodies to Detect ER Stress Markers 2.2 Quantitative RT-PCR (qPCR) Analysis 2.2.1 RNA Isolation 2.2.2 cDNA Synthesis 2.2.3 qPCR 2.3 Microscopic Analysis of ER structures 2.3.1 Transmission Electron Microscopy (TEM) 2.3.2 Fluorescence Microscopy 3 Methods 3.1 Western Blot Analysis 3.2 Quantitative RT-PCR (qPCR) 3.2.1 RNA Isolation 3.2.2 Reverse Transcription (cDNA Synthesis) 3.2.3 qPCR 3.3 Transmission Electron Microscopy (TEM) 3.4 Fluorescence Microscopy 4 Notes References Chapter 18: Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition 1 Introduction 2 Materials 2.1 Metabolic Flux Analysis 2.1.1 Cell Mito Stress Test 2.1.2 Energy Phenotype Test 2.1.3 Glycolysis Stress Test 2.2 Cell Number Measurement 2.2.1 Crystal Violet Staining 2.2.2 DAPI Staining of Cell Nuclei 2.3 Fluorogenic Assays 2.3.1 Flow Cytometric Analysis Accumulation of TMRE in Mitochondria Accumulation of MitoTracker in Mitochondria 2.3.2 Fluorescence Microscopic Analysis of Mitochondria After MitoTracker Staining 2.4 Western Blot Analysis 2.4.1 Preparation of Whole Cell Extracts (WCEs) 2.4.2 Determination of Protein Concentration 2.4.3 Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) 2.4.4 Western Blotting and Immunodetection 3 Methods 3.1 Metabolic Flux Analysis 3.1.1 Cell Mito Stress Test 3.1.2 Energy Phenotype Test 3.1.3 Glycolysis Stress Test 3.2 Cell Number Measurement 3.2.1 Crystal Violet Staining 3.2.2 DAPI Staining of Cell Nuclei 3.3 Fluorogenic Assays 3.3.1 Flow Cytometric Analysis 3.3.2 Fluorescence Microscopy of Mitochondria After MitotrackerTM Staining 3.4 Western Blot Analysis 3.4.1 Preparation of Whole-Cell Extracts 3.4.2 Determination of Protein Concentrations 3.4.3 SDS-PAGE 3.4.4 Western Blotting and Immunodetection 4 Notes References Chapter 19: Assessing the Effect of Histone Deacetylase Inhibitors on DNA Double-Strand Break Repair by Nonhomologous End Join... 1 Introduction 2 Materials 3 Methods 3.1 Culture and Transfection 3.2 Flow Cytometry 3.3 Influence of HDAC1, HDAC2, and HDAC3 on NHEJ 4 Notes References Chapter 20: Assessing SIRT7 Activity In Vivo and In Vitro in Response to DNA Damage 1 Introduction 2 Materials 2.1 Whole-Cell Extraction 2.2 Chromatin Fractionation 2.3 Immunoprecipitation Assay 2.4 In Vivo Deacetylation Assay 2.5 In Vitro Deacetylation Assay 2.6 In Vitro Desuccinylation Assay 3 Methods 3.1 Whole-Cell Extraction 3.2 Chromatin Fractionation 3.3 Immunoprecipitation Assay for Endogenous SIRT7 3.4 Immunoprecipitation Assay for FLAG-Tag Proteins 3.5 In Vivo Deacetylation Assay 3.6 In Vitro Deacetylation Assay 3.6.1 Transfection, Treatment, and Purification of FLAG-Tag Protein 3.6.2 Elution of FLAG-Tag Proteins (See Note 11) 3.6.3 In Vitro Deacetylation Reaction 3.7 In Vitro Desuccinylation Assay 4 Notes References Chapter 21: Methods to Evaluate the Effects of HAT/KAT Inhibition on SIAH2-Driven Reactive Oxygen Species Generation in Helico... 1 Introduction 2 Materials 2.1 Bacterial Culture 2.2 Cell Culture and Treatments 2.3 Site-Directed Mutagenesis (SDM), Cloning and Stable Cell Generation 2.4 Cell Harvesting and Protein Extraction 2.5 SDS-PAGE 2.6 Immunoblotting/Western Blotting 2.7 Immunofluorescence Microscopy 3 Methods 3.1 Cell Culture 3.2 H. pylori Culture and Infection 3.3 Cell Transfection and Stable Cell Generation 3.4 CTK7A Treatment and H. pylori Infection 3.5 Cell Harvest and Lysate Preparation 3.6 SDS-PAGE 3.6.1 Composition of the Resolving Gel Solution 3.6.2 Composition of the Stacking Gel Solution 3.6.3 Gel Casting 3.7 Immunoblotting/Western Blotting 3.8 ROS Detection by Immunofluorescence Microscopy 4 Notes References Chapter 22: Monitoring Changes in Intracellular Reactive Oxygen Species Levels in Response to Histone Deacetylase Inhibitors 1 Introduction 2 Materials 2.1 Cell Culture 2.2 ROS Measurement 3 Methods 3.1 Cell Culture and Harvesting of Cells 3.2 CM-H2DCFDA Staining 3.3 Statistical Analysis 4 Notes References Chapter 23: Single-Cell Analysis of Histone Acetylation Dynamics at Replication Forks Using PLA and SIRF 1 Introduction 2 Materials 2.1 Equipment 2.2 Cell Culture 2.3 Immunofluorescence 2.4 Primary Antibodies 2.5 Critical Commercial Assays* 3 Methods 3.1 Quantitative Detection of Protein Interactions and Modifications at Replication Forks 3.2 SIRF Assay in siRNA-Treated Cells for Protein Knockdown Analyses With and Without Fork Stalling 4 Notes References Chapter 24: Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction 1 Introduction 2 Materials 2.1 Cell Transfection 2.2 Cell Fixation and Staining 2.3 Epifluorescence Microscopy, Imaging, and Data Analysis 3 Methods 3.1 Cell Transfection 3.1.1 One Day Prior to Transfection 3.1.2 On the Day of Transfection 3.2 Cell Fixation and Staining 3.2.1 GFP Detection 3.2.2 Immunostaining Combined with GFP Detection 3.3 Epifluorescence Microscopy, Imaging, and Data Analysis 3.3.1 Image Acquisition 3.3.2 Image Analysis 3.3.3 Manual Measurement of GFP Areas 3.3.4 Automatic Measurement of GFP Areas 3.4 Statistical Data Analysis 4 Notes References Part IV: Modern Assays to Evaluate HDAC Functions and HDACi, Including CRISPR-Cas9 Techniques Chapter 25: A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Vir... 1 Introduction 2 Materials 2.1 Equipment 2.2 Tissue Culture 2.3 Plasmids 2.4 Library Plasmid Pool Amplification 2.5 Lentiviral Production and Transduction (Ready-to-Use Solutions) 2.6 NGS Library Preparation 2.7 Screening Analysis 3 Methods 3.1 Preparation of the Lentiviral sgRNA Libraries (Timing: 1-2 Weeks) 3.2 Optimization of the Screening Conditions (Timing: 8-10 Weeks) 3.3 Genome-Wide Screening for Viral Restriction Factors (Timing: 12-14 Weeks) 3.4 Sequencing of the NGS Libraries (Timing: 3-4 Weeks) 3.5 Screening Data Analysis (Timing: 1-2 Weeks) 3.6 Validation of the Top Candidate Hits (Timing: 5-6 Weeks) 4 Notes References Chapter 26: Cloning Strategy for HDAC1/HDAC2 Hybrid Protein Expression in Mammalian Cells 1 Introduction 2 Materials 2.1 Oligonucleotides 2.2 Polymerase Chain Reaction 2.3 Vector Restriction and Dephosphorylation of Vector 2.4 Agarose Gel Electrophoresis 2.5 DNA Restriction of PCR Product 2.6 Ligation 2.7 Transformation 2.8 DNA Isolation (Mini) 2.9 Sequencing 3 Methods 3.1 Design of Oligonucleotides 3.2 PCR of Inserts 3.3 Vector Restriction and Dephosphorylation 3.4 Agarose Gel Electrophoresis 3.5 Restriction of PCR Product 3.6 Ligation 3.7 Transformation of Bacteria 3.8 DNA Isolation (Mini) 3.9 Sequencing 4 Notes References Chapter 27: Continuous Histone Deacylase Activity Assays 1 Introduction 2 Materials 2.1 Enzymes 2.2 Peptide Substrates 2.3 Compounds Modulating the Deacylase Activities 2.4 Buffers and Solutions 2.5 Other Equipment 3 Methods 3.1 Assaying HDAC Activity 3.1.1 HDAC Activity Measurement for HDAC4 and Peptide B as Substrate 3.1.2 Determining a v/[S]-Plot for HDAC4 and Peptide B as Substrate 3.1.3 Peptide Calibration Line (Example: HDAC4 and Peptide B) 3.2 Determining IC50 Value (Example: HDAC11) 3.3 Fluorescence Displacement Assay for SIRT2 4 Notes References Chapter 28: CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells 1 Introduction 2 Materials 2.1 Plasmids, Cell Lines, and Bacteria 2.2 Buffer and Media 2.3 Consumables 2.4 Kits 2.5 Enzymes 2.6 Devices 3 Methods 3.1 Re-amplification and Sequencing of the sgRNA Libraries 3.1.1 Re-amplification 3.1.2 Sample Preparation 3.1.3 Sequencing 3.2 Cell Culture 3.2.1 Construction of CRISPRi/a Cell Lines 3.2.2 Validation of CRISPRi/a Cell Lines 3.2.3 Lentiviral sgRNA Library Transduction 3.2.4 Titration of the HDACi 3.3 HDACi Resistance Screening, Sample Preparation, and Sequencing 3.3.1 Screening 3.3.2 Sample Preparation 3.3.3 Sequencing 3.4 Bioinformatical Analysis of the Sequencing Results 3.5 Alternative Systems for Activation and Repression of Gene Expression 4 Notes References Chapter 29: Determining Potency of Inhibitors Targeting Histone Deacetylase 6 by Quantification of Acetylated Tubulin in Cells 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Sample Preparation 2.3 SDS-PAGE and Western Blots 2.3.1 Reagents 2.3.2 Primary Antibodies 2.3.3 Secondary Antibodies 2.4 Imaging System 3 Methods 3.1 Cell Culture, Treatment, and Harvesting 3.2 SDS-PAGE and Western Blotting 3.3 Probing of Western Blots 3.4 Quantification of Western Blot Signals Using the Typhoon FLA 9500 Imager and the Quantity One Image Analysis Software 3.5 Time Considerations and Expected Results 4 Notes References Chapter 30: Expression and Crystallization of HDAC6 Tandem Catalytic Domains 1 Introduction 2 Materials 2.1 Expression Construct Design and Cloning 2.2 Protein Expression 2.3 Protein Purification 2.4 Crystallization 3 Methods 3.1 Expression Construct Design and Cloning 3.2 Protein Expression 3.3 Protein Purification 3.4 Crystallization 4 Notes References Chapter 31: Fluorescence Polarization-Based Competition Assays to Evaluate Histone Deacetylase 6 Inhibitors 1 Introduction 2 Materials 2.1 Protein Purification 2.1.1 Equipment 2.1.2 Protein Purification Buffers 2.2 Fluorescence Polarization Displacement Assay 2.2.1 Equipment 2.2.2 Materials and Reagents (Fig. 3) 3 Methods 3.1 Protein Expression and Purification of zfHDAC6 CD2 3.2 Fluorescence Polarization Binding Assay Protocol 3.3 Fluorescence Polarization Competition Assay Protocol 4 Notes References Chapter 32: Immunoprecipitation of HDAC6 and Interacting Proteins 1 Introduction 2 Materials 2.1 Establishment and Characterization of Anti-mouse HDAC6-Specific Antibody 2.2 GFP-HDAC6 Transfection 2.3 MG132 and Cytochalasin D Treatment 2.4 Preparation of Cell Lysate for Immunoprecipitation and Immunoblotting 2.5 Immunoprecipitation of HDAC6 2.5.1 GFP-HDAC6 Preparation of Cell Lysate for Immunoprecipitation and Immunoblotting 2.5.2 Immunoprecipitation of GFP-HDAC6 2.6 Immunoblotting to Detect HDAC6 and Its Interacting Partners 2.7 Identification of HDAC6 Interacting Partners by Mass Spectrometry 3 Methods 3.1 Establishment and Characterization of Anti-HDAC6-Specific Antibody 3.2 GFP-HDAC6 Transfection 3.3 MG132 and Cytochalasin D Treatment 3.4 Preparation of Cell Lysate for Immunoprecipitation and Immunoblotting 3.4.1 MEF Cell Lysis with CSK Buffer 3.4.2 GFP-HDAC6 Transfected 293T Cell Lysis with CSK Buffer 3.5 Immunoprecipitation of HDAC6 3.5.1 Immunoprecipitation of Endogenous Mouse HDAC6 from MEF Cell Lysate 3.5.2 Immunoprecipitation of GFP-HDAC6 from 293T Cell Lysate 3.6 Immunoblotting to Detect HDAC6 and its Interacting Partners 3.6.1 Detection of Endogenous Mouse HDAC6 and Interacting Proteins 3.6.2 Detection of GFP-HDAC6 and Ub 3.7 Identification of HDAC6 Interacting Partners by Mass Spectrometry (MS) 4 Notes References Index