Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Cell Culture and Treatment
2.3. Measurement of Glutathione
2.4. MTT Assay
2.5. Measurement of Necrotic Cell Death
2.6. Quantitative Polymerase Chain Reaction Analysis
2.7. Measurement of Reactive Oxygen Species and Lipid Peroxidation
2.8. Immunoblot Analysis
2.9. Plasmid Construction, Transient Transfection, and Reporter Gene Assay
2.10. In Silico Analysis of BTG1 Promoter
2.11. Statistical Analysis
3. Results
3.1. Cystine and Methionine Deficiency Promotes Ferroptosis in Hepatocyte-Derived Cells
3.2. Cystine and Methionine Deficiency Activates ISR in Hepatocyte-Derived Cells
3.3. Cystine and Methionine Deficiency Induces BTG1 in an ATF4-Dependent Manner
3.4. BTG1 KO Prevents Ferroptosis Induced by Cystine and Methionine Deficiency
4. Discussion
5. 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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qPCR Analysis | ||||
Gene Symbol | Forward Primer | Backward Primer | Accession Number | Product Size (bp) |
ATF4 | 5′-GTTCTCCAGCGACAAGGCTA-3′ | 5′-TCTCCAACATCCAATCTGTCC-3′ | NM_001675.4 | 119 |
BTG1 | 5′-AGCGGATTGGACTGAGCAG-3′ | 5′-GGTGCTGTTTTGAGTGCTAC-3′ | NM_001731.2 | 161 |
CHOP | 5′-GCACCTCCCAGAGCCCTCACTCTCC-3′ | 5′-GTCTACTCCAAGCCTTCCCCCTGCG-3′ | NG_027674.1 | 422 |
GAPDH | 5′-GAAGGTGAAGGTCGGAGTC-3′ | 5′-GAAGATGGTGATGGGATTTC-3′ | NM_002046.4 | 226 |
GRP78 | 5′-TGCTTGATGTATGTCCCCTTA-3′ | 5′-CCTTGTCTTCAGCTGTCACT-3′ | NM_005347.5 | 303 |
PTGS2 | 5′-ATATGTTCTCCTGCCTACTGGA-3′ | 5′-GCCCTTCACGTTATTGCAGAT-3′ | NM_000963.3 | 108 |
SLC7A11 | 5′-TGCTGGGCTGATTTATCTTCG-3′ | 5′-GAAAGGGCAACCATGAAGAGG-3′ | NM_014331.4 | 114 |
TRIB3 | 5′-TGTCTTCGCTGACCGTGAGA-3′ | 5′-ACGCGTGCTTGTCCCACAGG-3′ | NM_021158.5 | 101 |
Construction of Recombinant Plasmids | ||||
Plasmid Name | Forward Primer | Backward Primer | ||
hpBTG1-luc | 5′-CTCGAGACCCTGTCTTAGGCCTAATCG-3′ | 5′-AGATCTTCCAGCTCCGCAGCATTCGAA-3′ | ||
mpBTG1-luc | 5′-CTCGAGGTGGTGTGTATTGCATCTGATGACC-3′ | 5′-AGATCTCACATCGCTCGGACCTCCCCAGCC-3′ | ||
* mpBTG1- ΔA4RE-luc | 5′-GCCTCGAGGTGGTGTGTGATGACCAACTAAACTC-3′ 5′-GGATGAGAGGGAGGTGCTTGTAAACAAATAAACCCCC-3′ | 5′-GAGTTTAGTTGGTCATCACACACCACCTCGAGGC-3′ 5′-GGGGGTTTATTTGTTTACAAGCACCTCCCTCTCATCC-3′ |
Species | Chromosomal Location | Matrix ID | Score | Relative Score | * Binding Site (bp) | ** Orientation | DNA Sequence |
---|---|---|---|---|---|---|---|
Homo sapiens | chr12:92,145,847–92,147,046 | MA0833.1 | 13.566 | 0.9104 | −197 to −185 | – | 5′-AGCTGACGTAATC-3′ |
MA0833.2 | 10.3809 | 0.8701 | −198 to −185 | – | 5′-AGCTGACGTAATCC-3′ | ||
MA0833.1 | 11.1549 | 0.8767 | −115 to −103 | – | 5′-TTGTGACGCTTGC-3′ | ||
Mus musculus | chr10:96,615,806–96,617,005 | MA0833.2 | 10.6486 | 0.8737 | −1013 to −1000 | – | 5′-ATCAGATGCAATAC-3′ |
MA0833.1 | 9.3852 | 0.8519 | −1012 to −1000 | – | 5′-ATCAGATGCAATA-3′ | ||
MA0833.1 | 13.566 | 0.9104 | −197 to −185 | – | 5′-AGCTGACGTAATC-3′ | ||
MA0833.2 | 11.1591 | 0.8806 | −198 to −185 | – | 5′-AGCTGACGTAATCT-3′ | ||
Rattus norvegicus | chr7:37,811,631–37,812,830 | MA0833.1 | 13.566 | 0.9104 | −516 to −504 | – | 5′-AGCTGACGTAATC-3′ |
MA0833.2 | 11.1591 | 0.8806 | −517 to −504 | – | 5′-AGCTGACGTAATCT-3′ |
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Cho, I.-J.; Kim, D.; Kim, E.-O.; Jegal, K.-H.; Kim, J.-K.; Park, S.-M.; Zhao, R.; Ki, S.-H.; Kim, S.-C.; Ku, S.-K. Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1. Antioxidants 2021, 10, 1543. https://doi.org/10.3390/antiox10101543
Cho I-J, Kim D, Kim E-O, Jegal K-H, Kim J-K, Park S-M, Zhao R, Ki S-H, Kim S-C, Ku S-K. Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1. Antioxidants. 2021; 10(10):1543. https://doi.org/10.3390/antiox10101543
Chicago/Turabian StyleCho, Il-Je, Doyeon Kim, Eun-Ok Kim, Kyung-Hwan Jegal, Jae-Kwang Kim, Sang-Mi Park, Rongjie Zhao, Sung-Hwan Ki, Sang-Chan Kim, and Sae-Kwang Ku. 2021. "Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1" Antioxidants 10, no. 10: 1543. https://doi.org/10.3390/antiox10101543