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LIVER Publication-derived

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Source Clavería-Cabello et al., 2023 · CIMA, University of Navarra, Pamplona, Spain · 10.1016/j.jhep.2023.05.031

👤 Alex Clavería-Cabello, Jose Maria Herranz, Maria Ujue Latasa, Maria Arechederra, Iker Uriarte, Carmen Berasain, Maite García Fernandez-Barrena, Matias Antonio Avila ⏱ 63 days 📋 16 phases 🧫 Primary HB cell lines, established HB cell lines (HuH6, HepT1, HepG2), patient-derived xenograft cells

Abstract

This protocol identifies dysregulated epigenetic genes in hepatoblastoma tissues and validates the histone methyltransferase G9a as a therapeutic target. Using transcriptomic analysis of 180 epigenetic genes, pharmacological targeting of G9a with CM272 and genetic mouse models, the study demonstrates that G9a inhibition suppresses hepatoblastoma growth by counteracting metabolic reprogramming mediated by c-MYC and ATF4.

Cell source
Primary HB cell lines, established HB cell lines (HuH6, HepT1, HepG2), patient-derived xenograft cells
Application
Disease modeling and drug screening in hepatoblastoma

Protocol overview

86 steps across 16 phases

Cell Culture and Sample Preparation Days 1-3
  1. 1 Primary hepatoblastoma cell line culture
  2. 2 Cell seeding for proliferation studies
  3. 3 Tissue sample collection and processing
In Vitro Drug Treatment and Viability Assessment Days 3-5
  1. 1 Drug treatment of cells
  2. 2 Cell viability measurement
  3. 3 GI50 calculation
  4. 4 Combination index (CI) calculation
Gene Expression Analysis (RNA extraction and qRT-PCR) Days 5-7
  1. 1 Total RNA extraction
  2. 2 Reverse transcription
  3. 3 Quantitative RT-PCR
Chromatin Immunoprecipitation (ChIP) Days 7-10
  1. 1 Chromatin crosslinking
  2. 2 Cell lysis and nuclear preparation
  3. 3 Nuclear washes
  4. 4 Chromatin sonication
  5. 5 Pre-clearing and antibody incubation
  6. 6 Bead recovery and washing
  7. 7 DNA elution and crosslink reversal
  8. 8 Final purification
  9. 9 ChIP-qPCR analysis
Histone Extraction and Immunoblot Analysis Days 10-12
  1. 1 Histone isolation
  2. 2 Histone precipitation and preparation
  3. 3 Histone quantification
  4. 4 Immunoblot sample preparation
  5. 5 Western blot detection
Colony Formation and Migration Assays Days 12-22
  1. 1 Colony formation assay setup
  2. 2 Colony fixation and staining
  3. 3 Colony documentation and analysis
  4. 4 Migration assay setup
  5. 5 Transwell fixation and staining
  6. 6 Migration quantification
Immunofluorescence Analysis Days 22-24
  1. 1 Cell culture and treatment
  2. 2 Cell fixation and permeabilization
  3. 3 DNA denaturation
  4. 4 Blocking and primary antibody incubation
  5. 5 Secondary antibody and mounting
  6. 6 Image acquisition
Organoid Culture and Viability Assessment Days 24-31
  1. 1 Organoid generation from patient tissue
  2. 2 Organoid culture in specialized medium
  3. 3 Weekly organoid passaging
  4. 4 Organoid drug treatment
  5. 5 Organoid viability assessment
Plasmid Transfection and Overexpression Studies Days 31-33
  1. 1 Plasmid preparation
  2. 2 G9a overexpression transfection
  3. 3 G9a overexpression verification and analysis
  4. 4 c-MYC overexpression transfection
  5. 5 c-MYC overexpression treatment and analysis
Mouse Xenograft Studies Days 33-63 (variable)
  1. 1 Subcutaneous tumor implantation
  2. 2 CM272 treatment administration
  3. 3 Tumor harvesting and processing
Generation and Analysis of G9a Knockout Mouse Model Days 63+ (variable)
  1. 1 Hepatocyte-specific G9a knockout mouse generation
  2. 2 Hydrodynamic tail-vein injection preparation
  3. 3 Hydrodynamic tail-vein injection
  4. 4 Post-injection monitoring and sample collection
Proteomic Analysis (LC-ESI-MS/MS) Days 33-37
  1. 1 Cell lysis and protein extraction
  2. 2 Sample homogenization and clarification
  3. 3 Protein quantification and reduction/alkylation
  4. 4 S-Trap protein digestion
  5. 5 Trypsin digestion on S-Trap
  6. 6 Peptide elution and cleanup
  7. 7 TMT 16plex labeling
  8. 8 Liquid chromatography setup and separation
  9. 9 Mass spectrometry data acquisition
  10. 10 Data analysis and protein identification
Metabolomic Analysis (UHPLC-MS) Days 37-40
  1. 1 Cell preparation for metabolite extraction
  2. 2 Metabolite extraction
  3. 3 Sample derivatization and preparation
  4. 4 UHPLC-MS analysis
  5. 5 Data processing and normalization
Metabolic Flux Analysis (Seahorse) Days 40-41
  1. 1 Cell seeding and CM272 treatment
  2. 2 Medium exchange and temperature equilibration
  3. 3 Basal OCR and ECAR measurement
  4. 4 Stress test with metabolic inhibitors
  5. 5 Data normalization and analysis
Immunohistochemical Analysis Days 41-44
  1. 1 Tissue section preparation
  2. 2 Antigen retrieval
  3. 3 Primary antibody incubation
  4. 4 Secondary antibody and detection
  5. 5 Counterstaining and mounting
  6. 6 Image acquisition and quantification
RNA Sequencing Days 44-50
  1. 1 RNA quality and quantity assessment
  2. 2 Library preparation
  3. 3 mRNA selection and fragmentation
  4. 4 Second-strand synthesis and cDNA preparation
  5. 5 Library amplification and quality control
  6. 6 High-throughput sequencing
  7. 7 Data archiving

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Clavería-Cabello et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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