Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing
Abstract
:1. Introduction
2. Results
2.1. PA200 Regulates Transcription of Genes Involved in the Key Cellular Processes
2.2. Mitochondrial Stress Assay Indicates Mitochondrial Dysfunction in shPA200 Cells
2.3. The Genetic Ablation of PA200 Results in Increased Glycolysis and Glycolytic Capacity
2.4. Preserved Long and Compact Tubular Mitochondrial Morphology in shPA200 Cells after Selective Mitochondrial Inhibitor Treatment
2.5. Effects of PA200 Knockdown on mRNA Expression of Genes Related to Mitochondrial Fusion and Fission
2.6. Genetic Ablation of PA200 Leads to Changes in Opa1 Processing in Cells Exposed to Selective Mitochondrial Insult
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Mitotracker Red CMXRos Staining for High-Content-Screening Confocal Microscopy (HCS)
4.3. Mitochondrial Morphology Analysis
4.4. RNA Extraction and Quantitative RT-PCR
4.5. Quantitative Real-Time PCR
4.6. Mitochondrial Fractionation
4.7. SDS-PAGE and Western Blot
4.8. Seahorse XF 96 Flux Analysis
4.8.1. Mitochondrial Stress Test Assay
4.8.2. Glycolysis Stress Test Assay
4.9. RNA-Seq
4.10. RNA-Seq Data Analysis
4.11. Functional Analysis of RNA-Seq Data
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Name | Forward Primer (5′–3′) | Reverse Primer (5′–3′) |
---|---|---|
hPSME4 | ATGGAGAGTGCCTGAACTATTG | GTAGGTCAGCACACTTCCTATTC |
hFIS1 | AGCTGGTGTCTGTGGAGGAC | ACGATGCCTTTACGGATGTC |
hMFN1 | CGGAACTTGATCGAATAGCC | AGAGCTCTTCCCACTGCTTG |
hMFN2 | ATGCATCCCCACTTAAGCAC | AGCACCTCACTGATGCCTCT |
hDNM1L | AGATCTCATCCCGCTGGTC | CAGATCCTCGAGGCAAGAAG |
hMIEF2 | GCAGAGTTCTCCCAGAAACG | GTCTGCCTTGGTGTCATCCT |
hMIEF1 | GCAAAGGCAAGAAGGATGAC | CTTCATGTCCCTGTTCAGCA |
hOPA1 | CACTTCCTGGGTCATTCCTG | TGCTTCGTGAAACCAGATGT |
hMFF | AAACGCTGACCTGGAACAAG | TTTTCAGTGCCAGGGGTTTA |
hβ-actin | GACCCAGATCATGTTTGAGACC | CATCACGATGCCAGTGGTAC |
Antibody | Source | Catalog Number | Host | Dilution |
---|---|---|---|---|
Drp1 | BD Biosciences | # 611112 | Mouse | 1:1000 |
Mfn1 | Abnova | # H00055669-M04 | Mouse | 1:1000 |
Mfn2 | Sigma Aldrich | # WH0009927M3 | Mouse | 1:800 |
Opa1 | Novus Biologicals | # NB110-55290 | Rabbit | 1:1000 |
OXPHOS | Abcam | # ab110413 | Mouse | 1:250 |
OMA1 | SantaCruz Biotechnology | # sc-515788 | Mouse | 1:500 |
YME1L | Proteintech | # 11510-1-AP | Rabbit | 1:1000 |
Fis1 | Invitrogen, Thermo Fisher | # PA1-41082 | Rabbit | 1:1000 |
Hsp60 | Invitrogen, Thermo Fisher | # MA3-012 | Mouse | 1:1000 |
β-actin-HRP | SantaCruz Biotechnology | # sc-1616 | Goat | 1:5000 |
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Douida, A.; Batista, F.; Boto, P.; Regdon, Z.; Robaszkiewicz, A.; Tar, K. Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing. Int. J. Mol. Sci. 2021, 22, 1629. https://doi.org/10.3390/ijms22041629
Douida A, Batista F, Boto P, Regdon Z, Robaszkiewicz A, Tar K. Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing. International Journal of Molecular Sciences. 2021; 22(4):1629. https://doi.org/10.3390/ijms22041629
Chicago/Turabian StyleDouida, Abdennour, Frank Batista, Pal Boto, Zsolt Regdon, Agnieszka Robaszkiewicz, and Krisztina Tar. 2021. "Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing" International Journal of Molecular Sciences 22, no. 4: 1629. https://doi.org/10.3390/ijms22041629