Hepatocyte Thorns, A Novel Drug-Induced Stress Response in Human and Mouse Liver Spheroids
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. ATP Assay
2.3. Thorn Isolation
2.4. Deglycosylation and Glycosylation Staining
2.5. Whole Spheroid Staining
2.6. RT-qPCR
2.7. Proteomics
2.7.1. Qualitative Proteomics
2.7.2. Quantitative Proteomics
2.7.3. Proteome Integral Solubility Alteration (PISA) Analysis
2.8. Scanning Electron Microscopy
2.9. Animal Experiments
2.10. Masson’s Trichrome Staining
3. Results
3.1. Chronic Drug Treatment Causes the Appearance of Thorn-Like Structures in Spheroid 3D Liver Model
3.2. Thorns Are Composed of Glycosylated Protein Fibres
3.3. Thorns Form Due to Lipid Metabolism Disturbances and Their Creation Is Stimulated by Reactive Oxygen Species
3.4. Thorns Represent Chronic Drug Treatment-Induced Hepatocyte Stress Response
3.5. Chronically GW6471-Treated Mice Exhibit Fibre Accumulation in the Liver
4. Discussion
4.1. Thorn Characteristics
4.2. Proposed Mechanisms of Thorn Formation
4.3. Comparable Effects of GW6471 in Mice
4.4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Identified Proteins | Sum of MK886 | Sum of DMSO |
---|---|---|
Spectrin alpha chain, non-erythrocytic 1 | 355 | 17 |
Cytoplasmic dynein 1 heavy chain 1 | 331 | 5 |
Filamin-A | 327 | 10 |
Spectrin beta chain, non-erythrocytic 1 | 319 | 32 |
CK, type I cytoskeletal 18 | 297 | 24 |
Filamin-B | 276 | 7 |
Clathrin heavy chain 1 | 266 | 18 |
Plectin | 261 | 3 |
Fatty acid synthase | 223 | 9 |
Talin-1 | 213 | 8 |
DNA-dependent protein kinase catalytic subunit | 207 | 1 |
Elongation factor 1-alpha 1 | 202 | 19 |
Tubulin beta-2B chain | 201 | 16 |
Tubulin beta-4A | 191 | 17 |
Ras GTPase-activating-like protein IQGAP2 | 186 | 7 |
Neuroblast differentiation-associated protein AHNAK | 175 | 9 |
Ras GTPase-activating-like protein IQGAP1 | 147 | 1 |
Aldo-keto reductase family 1 member C2 | 144 | 7 |
eIF-2-alpha kinase activator GCN1 | 128 | 2 |
Complement C4-B | 121 | 3 |
Elongation factor 2 | 118 | 3 |
Fructose-bisphosphate aldolase B | 112 | 11 |
Alpha-enolase | 108 | 8 |
Ribosome-binding protein 1 | 104 | 5 |
UTP--glucose-1-phosphate uridylyltransferase | 104 | 3 |
NADPH--cytochrome P450 reductase | 103 | 10 |
Cytochrome P450 2C8 | 101 | 10 |
Cytosolic 10-formyltetrahydrofolate dehydrogenase | 101 | 4 |
Heat shock protein HSP 90-alpha | 96 | 9 |
Tubulin beta chain | 241 | 41 |
CK, type II cytoskeletal 7 | 114 | 16 |
Identified Proteins | Sum of MK886 | Sum of DMSO |
---|---|---|
Filamin-A | 480 | 26 |
CK, type I cytoskeletal 18 | 424 | 42 |
Cytoplasmic dynein 1 heavy chain 1 | 420 | 6 |
Plectin | 397 | 3 |
Filamin-B | 393 | 15 |
Talin-1 | 308 | 12 |
Fatty acid synthase | 307 | 12 |
Tubulin beta-2B chain | 266 | 16 |
Ras GTPase-activating-like protein IQGAP2 | 261 | 25 |
DNA-dependent protein kinase catalytic subunit | 252 | 1 |
Elongation factor 1-alpha 1 | 239 | 24 |
Tubulin beta-4A chain | 235 | 17 |
CK, type II cytoskeletal 7 | 216 | 18 |
Ras GTPase-activating-like protein IQGAP1 | 205 | 5 |
Aldo-keto reductase family 1 member C2 | 182 | 12 |
Complement C4-B | 167 | 16 |
Elongation factor 2 | 163 | 6 |
Ribosome-binding protein 1 | 159 | 5 |
UTP--glucose-1-phosphate uridylyltransferase | 157 | 10 |
Alpha-enolase | 156 | 10 |
Coatomer subunit alpha | 153 | 1 |
eIF-2-alpha kinase activator GCN1 | 153 | 5 |
Formimidoyltransferase-cyclodeaminase | 141 | 13 |
C-1-tetrahydrofolate synthase, cytoplasmic | 140 | 8 |
Gelsolin | 134 | 9 |
Staphylococcal nuclease domain-containing protein 1 | 133 | 1 |
Cytosolic 10-formyltetrahydrofolate dehydrogenase | 133 | 4 |
Aldo-keto reductase family 1 member C3 | 121 | 9 |
Heat shock 70 kDa protein 1A | 113 | 6 |
Long-chain-fatty-acid--CoA ligase 5 | 112 | 8 |
Alpha-actinin-1 | 105 | 7 |
T-complex protein 1 subunit beta | 100 | 2 |
Glutamine--fructose-6-phosphate aminotransferase 1 | 99 | 3 |
ATP-dependent RNA helicase A | 96 | 1 |
Common Proteins | Cytoskeleton | Filament |
---|---|---|
Cytoplasmic dynein 1 heavy chain 1 | ✓ | ✓ |
Filamin-A | ✓ | ✓ |
CK, type I cytoskeletal 18 | ✓ | ✓ |
Filamin-B | ✓ | ✓ |
Plectin | ✓ | ✓ |
Fatty acid synthase | ||
Talin-1 | ✓ | |
DNA-dependent protein kinase catalytic subunit | ||
Elongation factor 1-alpha 1 | ||
Tubulin beta-2B chain | ✓ | ✓ |
Tubulin beta-4A | ✓ | ✓ |
Ras GTPase-activating-like protein IQGAP2 | ||
Neuroblast differentiation-associated protein AHNAK | ||
Ras GTPase-activating-like protein IQGAP1 | ✓ | |
Aldo-keto reductase family 1 member C2 | ||
eIF-2-alpha kinase activator GCN1 | ||
Complement C4-B | ||
Elongation factor 2 | ||
CK, type II cytoskeletal 7 | ✓ | ✓ |
Alpha-enolase | ||
UTP--glucose-1-phosphate uridylyltransferase | ||
Cytosolic 10-formyltetrahydrofolate dehydrogenase |
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Pridgeon, C.S.; Bolhuis, D.P.; Milosavljević, F.; Manojlović, M.; Végvári, Á.; Gaetani, M.; Jukić, M.M.; Ingelman-Sundberg, M. Hepatocyte Thorns, A Novel Drug-Induced Stress Response in Human and Mouse Liver Spheroids. Cells 2022, 11, 1597. https://doi.org/10.3390/cells11101597
Pridgeon CS, Bolhuis DP, Milosavljević F, Manojlović M, Végvári Á, Gaetani M, Jukić MM, Ingelman-Sundberg M. Hepatocyte Thorns, A Novel Drug-Induced Stress Response in Human and Mouse Liver Spheroids. Cells. 2022; 11(10):1597. https://doi.org/10.3390/cells11101597
Chicago/Turabian StylePridgeon, Chris S., Dian P. Bolhuis, Filip Milosavljević, Marina Manojlović, Ákos Végvári, Massimiliano Gaetani, Marin M. Jukić, and Magnus Ingelman-Sundberg. 2022. "Hepatocyte Thorns, A Novel Drug-Induced Stress Response in Human and Mouse Liver Spheroids" Cells 11, no. 10: 1597. https://doi.org/10.3390/cells11101597