Preface
Contents
Contributors
Chapter 1: Determination of CHK1 Cellular Localization by Immunofluorescence Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture and Treatment
3.2 Immunofluorescence Microscopy
4 Notes
References
Chapter 2: Detection of Post-translationally Modified p53 by Western Blotting
1 Introduction
2 Materials
2.1 Standard SDS-PAGE
2.2 Western Blot Transfer
2.3 Western Blot Development
3 Methods
3.1 Protein Extraction and Sample Preparation for SDS-PAGE
3.2 SDS-PAGE (See Note 1)
3.3 Western Blot Transfer
3.4 Western Blot Development
4 Notes
References
Chapter 3: Global Analyses to Identify Direct Transcriptional Targets of p53
1 Introduction
2 Materials
2.1 Cell Culture and Activation of p53 by Nutlin Treatment
2.2 Isolation of Nuclei for GRO-Seq
2.3 Nuclear Run-On and Nascent RNA Enrichment for GRO-Seq
2.4 Sequencing Library Preparation for GRO-Seq
2.5 Quantification and Pooling of GRO-Seq Libraries for Multiplex Sequencing
2.6 Cross-Linking and Whole-Cell Lysate Preparation for ChIP-Seq
2.7 Immunoprecipitation and DNA Purification for ChIP-Seq
2.8 Size Selection and Sequencing Library Preparation for ChIP-Seq
2.9 Quantification and Pooling of ChIP-Seq Libraries for Multiplex Sequencing
2.10 Isolation of Total Cellular RNA for RNA-Seq
2.11 Poly(A) Enrichment and Sequencing Library Preparation for RNA-Seq
2.12 Computational Tools
3 Methods
3.1 Cell Culture and Non-genotoxic Activation of p53
3.2 Profiling the Transcriptional Response to p53 Activation by Global Run-on Sequencing (GRO-Seq)
3.2.1 Isolation of Nuclei for GRO-Seq
3.2.2 Nuclear Run-on and RNA Extraction
3.2.3 Nascent RNA Enrichment and Sequencing Library Preparation for GRO-Seq
3.2.4 GRO-Seq Data Analysis and Visualization
3.3 Determining Genomic Binding Locations for p53 by Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
3.3.1 Formaldehyde Cross-Linking and Whole-Cell Extract Preparation
3.3.2 p53 Chromatin Immunoprecipitation and DNA Purification
3.3.3 p53 ChIP-Seq Data Analysis and Visualization
3.4 Measuring Steady-State Transcriptome Response to p53 Activation by RNA Sequencing (RNA-Seq)
3.4.1 Cell Harvest and Total RNA Extraction for RNA-Seq
3.4.2 Enrichment of mRNA by Poly(A) Selection
3.4.3 RNA-Seq Data Analysis and Visualization
4 Notes
References
Chapter 4: Analysis of Replication Dynamics Using the Single-Molecule DNA Fiber Spreading Assay
1 Introduction
2 Materials
2.1 Nucleotide Analogs
2.2 Lysis Buffer
2.3 Fixation Solution
2.4 Storage and Cleaning Solution for Glass Slides
2.5 Glass Slides and Coverslips
2.6 Solutions for Immunofluorescence Staining
2.7 Image Acquisition and Analysis
3 Methods
3.1 In Vivo Labeling of Nascent DNA: Incorporation of Halogenated Thymidine Analogs into Adherently Growing Cells
3.2 In Vivo Labeling of Nascent DNA: Incorporation of Halogenated Thymidine Analogs into Suspension Culture Cells
3.3 Spreading of the Pulse-Labeled DNA on Glass Slides
3.4 Immunofluorescence Staining of Halogenated Thymidine Analogs Incorporated into Nascent DNA
3.5 Image Acquisition of the DNA Fibers
3.6 Image Analysis of DNA Fibers
4 Notes
References
Chapter 5: Measuring Translation Efficiency by RNA Immunoprecipitation of Translation Initiation Factors
1 Introduction
2 Materials
3 Methods
3.1 Lysate Preparation
3.2 Immunoprecipitation
3.3 RT-PCR
4 Notes
References
Chapter 6: DNA Affinity Purification: A Pulldown Assay for Identifying and Analyzing Proteins Binding to Nucleic Acids
1 Introduction
2 Materials
2.1 Nuclei Extraction Buffer
2.2 Nuclei Lysis Buffer
2.3 Washing Buffer
2.4 Elution Buffer
3 Methods
3.1 Preparation of Biotinylated Double-Stranded DNA Probes
3.2 DNA Affinity Purification
3.3 Protein Analysis
4 Notes
References
Chapter 7: A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer
1 Introduction
2 Materials
2.1 Preparation of Ub, E1, and E2 Cdc34 (See Note 1)
2.2 Preparation of E3 ROC1-CUL1 CTD Complex
2.3 Preparation of Fluorescently Labeled Ub
2.4 FRET K48 di-Ub Assay
2.5 Running the Gel
2.6 Fluorescence Imaging
3 Methods
3.1 Preparation of Ub, E1, or E2 Cdc34
3.2 Preparation of E3 ROC1-CUL1 CTD Complex
3.3 Preparation of Fluorescently Labeled Ub
3.4 FRET K48 di-Ub Assay
3.5 Running the Gel
3.6 Fluorescence Imaging of the Gel
4 Notes
References
Chapter 8: DNA Damage Response in Xenopus laevis Cell-Free Extracts
1 Introduction
2 Materials
2.1 Animals
2.2 Hormones to Prime Frogs
2.3 General Equipment
2.4 CSF Extract
2.5 Activated CSF Extract/Interphase Extract I
2.6 ELB Extract/LSS Extract
2.7 Interphase Extract II
2.8 High-Speed Supernatant (HSS)
2.9 Nucleoplasmic Extract (NPE)
2.10 Immunodepletion of Extracts to Study Protein Function
2.11 Demembranated Sperm Chromatin
2.12 Generation of Double-Strand Breaks (DSB) in Chromatin
2.13 Preparation of plasmids containing Interstrand Cross-Links (ICLs)
2.14 Preparation of DNA with Double-Strand Breaks
2.15 Preparation of Biotinylated Substrates
2.16 DNA Damage Checkpoint Induced by Single-Strand DNA Gaps in Chromatin
2.17 DNA Damage Checkpoint Induced by DSB in Chromatin
2.18 Assay to Study Checkpoint Induced by ICLs
2.19 Study of the Checkpoint Induced by DNA Damage In Trans on DNA Replication
2.20 Phosphorylated Histone H2AX Detection (Endogenous)
2.21 Histone H2AX Phosphorylation Assay (Exogenous Substrate)
2.22 Phosphorylation of ATM/ATR Target Proteins
2.23 Chromatin Binding Assay
2.24 Binding Assay Using HSS/NPE and ICL Plasmid
3 Methods
3.1 Preparation of Extracts
3.1.1 CSF Extract
3.1.2 Activated CSF Extract/Interphase Extract I
3.1.3 ELB Extract/LSS Extract
3.1.4 Interphase Extract II
3.1.5 High-Speed Supernatant (HSS)
3.1.6 Nucleoplasmic Extract (NPE)
3.1.7 Immunodepletion of Extracts to Study Protein Function
3.2 Preparation of DNA Templates
3.2.1 Demembranated Sperm Chromatin
3.2.2 Generation of Double-Strand Breaks (DSBs) in Chromatin
3.2.3 Preparation of Plasmids containing Interstrand Cross-Links (ICLs)
3.2.4 Preparation of DNA with Double-Strand Breaks
3.2.5 Preparation of Biotinylated Substrates
3.3 Use of the Xenopus System to Study Cell Cycle Checkpoints/Checkpoint Assays
3.3.1 DNA Damage Response Induced by Single-Strand DNA Gaps in Chromatin
3.3.2 DNA Damage Responses Induced by DSB in Chromatin
3.3.3 Assay to Study Checkpoint Induced by ICL
3.3.4 Study of the Checkpoint Induced by DNA Damage In-Trans on DNA Replication
3.3.5 Study of Phosphorylation to Assess Checkpoint Activation
3.3.6 Phosphorylated Histone H2AX Detection (Endogenous Substrate)
3.3.7 Histone H2AX Phosphorylation Assay (Exogenous Substrate)
3.3.8 Phosphorylation of ATM/ATR Target Proteins
3.3.9 Chromatin Binding Assay
3.3.10 Binding Assay Using HSS/NPE and ICL Plasmid
4 Notes
References
Chapter 9: Mammalian Cell Fusion Assays for the Study of Cell Cycle Progression by Functional Complementation
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Medium, Buffers, and Supplements
2.3 Plastics and Equipment
2.4 Chemical Reagents
2.5 Immunofluorescence
3 Methods
3.1 Cell Synchronization
3.1.1 Synchronization of HeLa Cells at the G1/S Transition by Double Thymidine/Mimosine Block
3.1.2 Synchronization of Cells in Mitosis or Early G1
3.2 Cell-Cell Fusion and Heterokaryon Formation
3.3 Analysis of Heterokaryons by Immunofluorescence
4 Notes
References
Chapter 10: Knockdown of Target Genes by siRNA In Vitro
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 11: Assaying Cell Cycle Status Using Flow Cytometry
1 Introduction
2 Materials
2.1 Analysis of Cell Cycle Status by Costaining for DNA Content and Ki-67
2.1.1 Solutions and Reagents
2.1.2 Special Equipment
2.2 Staining of Cell Surface Markers and for Ki-67/PI
2.2.1 Solutions and Reagents
2.2.2 Special Equipment
2.3 Analysis of Cell Cycle by Hoechst 33342 and Pyronin Y Staining
2.3.1 Solutions and Reagents
2.3.2 Special Equipment
2.4 Analysis of Cell Cycle Using BrdU Incorporation-Based Method
2.4.1 Solutions and Reagents
2.5 Analysis of Cell Cycle Using EdU Incorporation-Based Method
2.5.1 Solutions and Reagents
2.5.2 Prepare Stock Solutions (See Note 7)
3 Methods
3.1 Analysis of Cell Cycle Status by Costaining for DNA Content and Ki-67
3.1.1 Harvest, Fix, and Permeabilize Cells Using 70% Cold Ethanol
3.1.2 Stain Cells with Ki-67 Antibody and Fluorescent DNA Dye
3.2 Staining of Cell Surface Markers and for Ki-67/PI
3.2.1 Stain Cell Surface Antigens with Fluorophore-Conjugated Antibodies
3.2.2 Fix and Permeabilize Cells for Intracellular Staining
3.2.3 Stain Cells with Ki-67 Antibody and Fluorescent DNA Dye
3.3 Analysis of Cell Cycle by Hoechst 33342 and Pyronin Y Staining
3.4 Analysis of Cell Cycle Using BrdU Incorporation-Based Method
3.5 Analysis of Cell Cycle Using EdU Incorporation-Based Method
3.5.1 EdU Incorporation Procedure
3.5.2 Cell Fixation and Permeabilization
3.5.3 EdU Detection (the ``Click Reaction´´)
3.6 Flow Cytometry
3.7 Dye Dilution Proliferation Assay
4 Notes
References
Chapter 12: Using TUNEL Assay to Quantitate p53-Induced Apoptosis in Mouse Tissues
1 Introduction
2 Materials
2.1 Drug Treatment
2.2 Fixation of Mouse Organs
2.3 Wash and Permeabilization
2.4 TUNEL Assay
2.5 Equipment
3 Methods
3.1 DNA Damage with Ionizing Radiation
3.2 DNA Damage with Doxorubicin
3.3 Fixation of Mouse Organs
3.4 Sectioning of Mouse Organs
3.5 TUNEL Assay
3.6 Acquisition of Image
4 Notes
References
Chapter 13: Generation and Analysis of dsDNA Breaks for Checkpoint and Repair Studies in Fission Yeast
1 Introduction
2 Materials
2.1 Electrophoretic Detection of Processed DSBs
2.2 Induction of DNA Double-Stranded Breaks and Microscopic Visualization of Sites of Homologous Recombination
2.3 Induction of DNA Double-Stranded Breaks and Processing of Lesions
2.4 Measuring Protein Recruitment to DNA Double-Strand Break Via Chromatin Immunoprecipitation (ChIP)
3 Methods
3.1 Electrophoretic Detection of Processed DSBs
3.2 Detection of Sites of Homologous Recombination Through Rad51 Immunofluorescence
3.3 Induction of a DNA Double-Stranded Break and Processing of Lesions
3.3.1 Induction of DNA Double-Strand Break (DSB)
3.3.2 DNA Purification (This Method Is Also Used for the Electrophoretic Analysis Outlined in Subheading 3.1)
3.3.3 Measuring Efficiency of Break Via qPCR
3.3.4 Measuring Resection Length Via qPCR
DNA Digestion
3.3.5 Analysis
3.4 Measuring Protein Recruitment to DNA Double-Strand Break Via Chromatin Immunoprecipitation (ChIP)
3.4.1 Preparing Dynabeads
3.4.2 ChIP Washes/Elution
3.4.3 Analysis (See Note 9)
4 Notes
References
Chapter 14: Calreticulin Exposure in Mitotic Catastrophe
1 Introduction
2 Materials
2.1 Disposable
2.2 Equipment
2.3 Reagents
3 Methods
3.1 Cell Culture
3.2 Drug Preparation
3.3 Cell Seeding
3.4 Immunostaining
3.5 Cytometer Preparation
4 Notes
References
Chapter 15: Quantification of eIF2Ξ± Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy
1 Introduction
2 Materials
2.1 Disposables
2.2 Equipment
2.3 Reagents
3 Method
4 Notes
References
Chapter 16: Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe
1 Introduction
2 Materials
2.1 Disposables
2.2 Equipment
2.3 Reagents
2.3.1 Culture
2.3.2 Treatments with Mitotic Catastrophe and Apoptotic Inducers
2.3.3 Clonogenic Assays
3 Methods
3.1 Cell Culture
3.2 Classical Clonogenic Assay
3.3 Fluorescent Clonogenic Assay Microscopy
3.4 Mitotic Catastrophe Analysis
4 Notes
References
Index