دانلود کتاب Autophagy and Cancer: Methods and Protocols

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Preface
Contents
Contributors
Part I: Overview of the Field
Chapter 1: A Balance Between Autophagy and Other Cell Death Modalities in Cancer
1 Introduction
2 Autophagy in Disease Pathogenesis
2.1 Autophagy in Neurodegenerative Diseases
2.2 Autophagy in Cardiovascular Disease
2.3 Autophagy in Viral Infections
3 Cross Talk Between Autophagy and Other Cell Death Modalities
3.1 Autophagy-Dependent Cell Death (ADCD)
3.1.1 Autosis
3.1.2 Ferroptosis
3.1.3 Entosis
3.2 Cross Talk Between Autophagy and Apoptosis
3.3 Cross Talk Between Autophagy and Necroptosis
4 Dual Role of Autophagy in Cancer
4.1 Autophagy as a Tumor Suppressor
4.2 Autophagy as a Tumor Promoter
5 Concluding Remarks
References
Part II: Chaperone-Mediated Autophagy (CMA)
Chapter 2: Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradi...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Subcellular Fractionation and Validation
3 Methods
3.1 Cell Seeding and Collection
3.2 Homogenization and Differential Centrifugations
3.3 Multilayered Density Gradient Centrifugations
3.4 Immunoblotting
4 Notes
References
Chapter 3: CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Human LAMP-2A Isoform-Specific CRISPR sgRNA Design
2.3 Crispr-Cas9 Mediated Genome Editing
2.4 Genotyping
3 Methods
3.1 Transfection of Cas9:sgRNA Ribonucleoprotein Complex
3.2 Detection of the Genomic Editing (Genotyping)
3.3 Single Clone Isolation and Selection for Genotypic and Phenotypic Characterization (Array Dilution Method) (See Note 15)
4 Notes
References
Part III: Macroautophagy
Chapter 4: Modified LC3 Dot Quantification Method
1 Introduction
2 Materials
3 Methods
3.1 Seeding the Cells, Transfection, and Immunofluorescence Staining of LC3
3.2 Imaging and Quantification
3.3 Calculating the Proportion of LC3 Intensity in Dots
4 Notes
References
Chapter 5: A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle
1 Introduction
1.1 Autophagy and Cancer
1.2 LC3-Based Methods for Measuring Autophagy
1.3 Method to Detect Autophagy Using LC3-II Staining by Flow Cytometry
2 Materials
2.1 Cell Culture
2.2 Sample Preparation and Staining
2.3 Equipment
3 Methods
3.1 Cell Seeding, Permeabilization, and Staining for LC3-II
3.2 Staining for DNA Content
3.3 Flow Cytometry and Data Analysis
4 Notes
References
Chapter 6: A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment
1 Introduction
2 Materials
2.1 Generation of the Reporter Construct
2.1.1 Oligonucleotides
2.1.2 PCR Amplification and Gel Purification
2.1.3 Generation of Entry-Clones by BP Recombination
2.1.4 Generation of Expression Vectors by LR Multisite Recombination
2.2 Virus Packaging
2.3 Target Cell Line Transduction
2.4 HiBiT-LC3 Measurements
2.5 Hoechst Staining
2.6 GFP-LC3 Measurements
2.7 siRNA and Plasmid Transfections
2.8 Protein Extraction and Western-Blot Analysis
3 Methods
3.1 Plasmid Generation
3.1.1 PCR Amplification of attB Constructs for BP Recombination
3.1.2 Generation of Entry-Clones by RP Recombination
3.1.3 Generation of Expression Clones by Multisite LR Recombination
3.2 Virus Packaging
3.3 Virus Transduction and Colony Selection
3.4 Testing of Stable Cell Line Clones
3.5 Example of Experimental Set Up
4 Notes
References
Chapter 7: Measuring Autophagic Cargo Flux with Keima-Based Probes
1 Introduction
2 Materials
2.1 Generation of Stable Cell Lines by Lentiviral Transduction
2.2 Assessing Autophagic Flux by Live-Cell Confocal Microscopy
2.3 Assessing Autophagic Flux by Flow Cytometry
2.4 Assessing Autophagic Flux by Western Blot Analysis of Keima Fusion-Protein Processing
2.5 Cells, Cell Culture Medium, and Cell Culture Plastic
2.6 Autophagy-Inducing and -Inhibiting Drugs
3 Methods
3.1 Generation of Stable Cell Lines by Lentiviral Transduction
3.2 Assessing Autophagic Flux by Live-Cell Confocal Microscopy
3.3 Assessing Autophagic Flux by Flow Cytometry
3.4 Assessing Autophagic Flux by Western Blot Analysis of mKeima Fusion-Protein Processing
4 Notes
References
Chapter 8: Detection of Nuclear Biomarkers for Chromosomal Instability
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Detection of CIN Biomarkers
2.3 Image Analysis
3 Methods
3.1 Cell Culture
3.2 Detection of CIN Biomarkers
3.3 Image Acquisition
3.4 Quantification of Micronuclei and Toroidal Nuclei
4 Notes
References
Chapter 9: Analysis of Autophagic Vesicles in Mitotic Cells
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
2.2.1 Equipment
2.2.2 Solutions
2.3 Image Analysis
3 Methods
3.1 Cell Culture
3.1.1 Preparation
3.1.2 Synchronization at Late G2 Phase of Cell Cycle
3.2 Immunofluorescence
3.3 Image Acquisition
3.4 Image Analysis with FIJI
3.4.1 Number and Size of Autophagic Vesicles
3.4.2 Intracellular Localization of Autophagic Vesicles
4 Notes
References
Chapter 10: Approaches and Protocols to Analyze Autophagy and Its Role in Death of Apoptosis-Resistant Senescent Tumor Cells
1 Introduction
2 Materials
2.1 Inhibitors
2.2 Antibodies
2.3 Plasmids
2.4 Reagents
2.5 Buffers and Solutions
2.5.1 Western Blotting
2.5.2 Immunocytochemistry
2.5.3 Actin Cytoskeleton Visualization
2.5.4 Annexin V-FITC Binding Analysis
2.5.5 Clonogenic Survival
2.5.6 Cell Cycle Analysis by FACS
2.5.7 Proteasome Activity Assay
2.5.8 DNA Gel Electrophoresis
2.5.9 Single Cell Gel Electrophoresis (Comet Assay)
2.5.10 Caspase Activity Assay
2.5.11 Senescence-Associated β-Galactosidase Detection
2.6 Consumables
2.7 Cell Culture
2.8 Equipment
2.9 Software
3 Methods
3.1 Analysis of the Autophagic Process
3.1.1 Analysis of LC3 I to II Conversion by Western Blot
3.1.2 Immunofluorescent Analysis of Autophagy by LC3 and LAMP1 Signal Colocalization
3.1.3 Analysis of Autophagy in Living Cells Using GFP-mRFP-LC3 Plasmid
3.1.4 p62/SQSTM1 Levels Analysis by Western Blot
3.2 Visualization and Analysis of Senescence Markers
3.2.1 Assessment Proliferation Activity in Senescent Cells
3.2.2 Analysis of Proliferation Activity by BRDU Pulse Labelling
3.2.3 Cell Cycle Arrest by FACS Analysis in Senescent Cells
3.2.4 Evaluation of Proliferation Arrest Based on Clonogenic Assay
3.2.5 Analysis of mTORC1 Activity for Evaluation of Senescence and Autophagy
3.2.6 Senescence-Associated β-Galactosidase Activity
3.2.7 Protein Accumulation in Senescent Cells
3.2.8 Actin Cytoskeleton
3.2.9 Novel Hallmark: Spatial Dissociation of Lysosomes and Autophagosomes
3.3 Analysis of Viability Upon Treatment with Autophagy Inhibitors and Signaling Pathways Kinase Inhibitors
3.3.1 Analysis of Cell Death Rate Using MTT Test
3.3.2 Estimation of Mitochondria Damage Using Mitochondria-Targeting Dyes
3.3.3 Assessment of Lysosomal Integrity
3.3.4 Proteasome Activity Assay
3.3.5 Assay for Detection of Caspase 3, 8, and 9 Pro-apoptotic Activities
3.3.6 Single Cell Gel Electrophoresis (Comet Assay) for Detection of DNA Strain Breaks
3.3.7 Detection of Apoptotic DNA Fragmentation by DNA Agarose Gel Electrophoresis
3.3.8 Detection of Phosphatidyl Serine on the Outer Membrane of Apoptotic Cells by Annexin V-FITC
3.3.9 Detection of Autophagic Cell Death and Necroptosis
4 Notes
References
Chapter 11: Concepts and Design of Introducing Synthetic MicroRNAs into Mammalian Cells
1 Introduction
2 Materials
2.1 In Vitro miRNA Transfection
2.2 Stem-Loop miRNA RT-qPCR
2.3 In Vivo miRNA Administration
3 Methods
3.1 In Vitro miRNA Transfection
3.1.1 Stem-Loop miRNA RT-qPCR on In Vitro Derived Material
3.1.2 Stem-Loop miRNA RT-qPCR on In Vivo Derived Material
3.2 In Vivo miRNA Administration
4 Notes
References
Chapter 12: Studying Autophagy Using a TMT-Based Quantitative Proteomics Approach
1 Introduction
1.1 Experimental Approaches to Study Autophagy
1.2 Experimental Design and MS Analysis Based on the Research Question
2 Materials
2.1 Cell Culture
2.2 Cell Lysate Preparation and Quantification
2.3 Protein Reduction and Alkylation
2.4 Protein Precipitation
2.5 Protein Digestion
2.6 Sample Purification or Clean-Up
2.7 TMT Labeling
2.8 Sample Fractionation
2.9 Mass Spectrometry
3 Methods
3.1 Experimental Design
3.2 Cell Culture
3.3 Sample Preparation for TMT-Based Mass Spectrometry
3.4 Sample Fractionation and TMT-Based Mass Spectrometry Analysis
3.5 Data Preprocessing and Statistical Analysis
3.6 Biological Analysis
3.7 Protein Network Interactions
3.8 LIR (LC3 Interacting Region) Motif-Containing Proteins (LIRCPs) Search Analysis
4 Notes
References
Part IV: Mitophagy
Chapter 13: Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy
1 Introduction
1.1 Experimental Context and Background
1.2 Object-Based Image Analysis (OBIA)
1.3 ROIs Based on Mitochondria
1.4 ROIs Based on Autophagic Foci
1.5 Image Segmentation and Processing
2 Materials
2.1 Mammalian Cell Culture and Reagents
2.2 Immunofluorescence
2.3 Imaging
2.4 Software
3 Method
3.1 Sample Preparation
3.2 Immunostaining
3.3 Optical Imaging
3.3.1 Microscope Setup and Configuration
3.3.2 Data Acquisition
3.4 Image Analysis
3.4.1 OBIA of Mitochondria
3.4.2 OBIA of Autophagic Foci
3.5 Numerical Analyses
3.5.1 Mitochondrial Recruitment of an Autophagy Receptor During Mitophagy
3.5.2 Average Size of Mitochondrially Associated ATG13 Foci During Mitophagy
4 Notes
References
Chapter 14: Induction and Detection of Mitophagy
1 Introduction
1.1 Mechanisms of Mitophagy
1.2 The Role of Mitophagy in Diseases
1.3 Mitophagy Induction
2 Materials
2.1 Assessment of Protein Migration to Mitochondria Upon Mitophagy Induction
2.2 Analysis of Proteins of Interest by Western Blotting
2.3 Analysis of Proteins of Interest Using Confocal Microscopy
2.4 Monitoring the Mitochondrial Population Using a Flow Cytometer
2.5 Mitochondrial Quality Control
3 Methods
3.1 Fractionation
3.2 Western Blotting (Fig. 2)
3.3 Confocal Microscopy
3.4 Flow Cytometry
3.5 Mitochondria Quality Control
4 Notes
References
Part V: Autophagy Analysis In Vivo
Chapter 15: Analysis of Pancreatic Acinar Protein Solubility in Autophagy-Deficient Mice
1 Introduction
2 Materials
3 Method
3.1 Rapid Isolation and Storage of Fresh Pancreatic Tissue
3.2 Cold Grinding of Samples to Powder
3.3 Processing Samples for Protein
3.4 Processing Samples for RNA
3.5 Analysis of Samples and Interpretation of Results
4 Notes
References
Chapter 16: Studying Autophagy In Vivo in the Mammary Gland and in Xenograft Samples
1 Introduction
2 Materials
2.1 Protein Extraction and Immunoprecipitation (IP) from Mammary Glands
2.2 GFP-LC3 Quantification on Mammary Glands
2.3 Protein Extraction and IP from Xenografts
2.4 Histopathological Analysis from Xenografts Tissue
2.5 Sample Preparation for RNAseq
2.6 Electron Microscopy (EM)
3 Methods
3.1 Protein Extraction and Bcl-2 IP from Mammary Glands
3.2 In Vivo Autophagy Assay: GFP-LC3 Analysis in Mammary Gland
3.3 Protein Extraction and IP from Xenografts
3.4 Histopathological Analysis from Xenografts Tissue
3.5 Sample Preparation for RNAseq
3.6 Electron Microscopy
4 Notes
References
Part VI: Computational Approaches
Chapter 17: A Method to Identify Potential Prognostic Markers Across Distinct Tumor Types
1 Introduction
1.1 Autophagy and Cancer
1.2 The Cancer Genome Atlas
1.3 Type of Data in the TCGA
1.4 Pipeline for RNA seq Data Analysis
1.5 Matching RNA seq Data with Clinical Data
2 Materials
2.1 Computer Programs
2.2 TCGA RNA-seq Dataset Retrieval
3 Methods
3.1 RNA-seq Count Data Normalization
3.2 Analysis of Ten Key Genes in Autophagy and Their Association to Overall Patient Survival
3.3 Clinical Dataset Preparation
3.4 Overall Survival Analysis Using R
3.5 Visualization
4 Notes
References
Chapter 18: A Method for Coexpression Analysis
1 Introduction
1.1 Autophagy and Cancer Metabolism
1.2 Cancer Cell Line Encyclopedia
2 Materials
2.1 R, Rstudio, and R Package
2.2 Data
3 Methods
3.1 Prepare the Data for the Analysis
3.2 Correlation Analysis
3.2.1 Correlation Between Two Variables
3.2.2 Correlations for All Variables
3.2.3 Correlation Coefficient Description
3.3 Visualizations
4 Notes
References
Part VII: Metabolism
Chapter 19: Methodological Approaches for Assessing Metabolomic Changes in Glioblastomas
Abbreviations
1 Introduction
2 Impact of Metabolism on Cell Death in GBM
2.1 Ferroptosis and Its Relationship to the Mitochondrial Respiratory Chain
2.2 Apoptosis, Bcl-2 Family Members, and BH3 Mimetics
2.3 Autophagy and Its Association with GBM Metabolism
3 Approaches to Dissect GBM Metabolism
3.1 Metabolic Imaging
3.2 Carbon Tracing Experiments, Involving U-13C-Labeled Metabolites
3.3 Extracellular Flux Analysis on the Seahorse Instrument
3.3.1 Mitochondrial Stress Assay on the Seahorse Analyzer
3.3.2 Glycolysis Stress Assay on the Seahorse Analyzer
3.3.3 Mitochondrial Fuel Utilization Assay
3.3.4 Endogenous and Exogenous Fatty Acid Oxidation Assay
4 Conclusions
References
Chapter 20: Extraction of Metabolites from Cancer Cells
1 Introduction
1.1 Role of Autophagy in Metabolism
1.2 Metabolome and Metabolomics
1.3 Extraction of Metabolites
2 Materials
2.1 Cell Line(s)
2.2 Cell Culturing Materials
2.3 Sample Extraction
2.4 Equipment
3 Methods
3.1 Preparation of the Cells
3.2 Preparation of Filter Units
3.3 Collecting the Samples
4 Notes
References
Chapter 21: Measuring Glucose Consumption and Gluconeogenesis in 3D Human Tissue Cultures with Nanoliter Input Volumes
1 Introduction
2 Materials
2.1 Cell Culture and Sample Retrieval (Fig. 2)
2.2 Assay Solutions and Reading
2.3 Reagent Preparation
2.4 Instrumentation and Logistics
3 Methods
3.1 Cell Culture
3.1.1 Extended Conditioning/Treatment Period
3.1.2 Retrieval of Samples for Glucose Measurement
3.2 Assay Run
3.3 Data Analysis
4 Notes
References
Index