Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Cell Culture
2.3. Transient Transfection
2.4. In Vitro Cell Proliferation Assay (SRB Assay)
2.5. Detection of Cell Death
2.6. Determination of Cellular ATP Level
2.7. MitoTracker Red Staining
2.8. Immunofluorescence
2.9. Total RNA Preparation and Reverse Transcription–Quantitative PCR (RT-qPCR)
2.10. SDS-PAGE and Western Blotting
2.11. Determination of Lipid Peroxidation (TBARS Assay)
2.12. Determination of the Oxygen Consumption Rate (OCR)
2.13. Measurement of Superoxide Production (DHE Staining)
2.14. Electron Microscopy
2.15. Statistical Analysis
3. Results
3.1. Silencing of PARP2 Leads to Fragmented Mitochondria
3.2. SIRT1 Activation, Mitophagy, mtUPR, and the Deregulation of the Mitochondrial Fusion–Fission System Are Not Involved in Mitochondrial Fragmentation in PARP2-Silenced Cells
3.3. Mitochondrial Fragmentation Is Not Specific for C2C12 Myoblasts
3.4. Silencing of PARP2 Leads to Oxidative Stress, Originating Partly from the Mitochondria through Mitochondrial Protein Imbalance and Fragmentation
3.5. PARP1 and PARP3 Are Not Involved in Mitochondrial Fragmentation Elicited by the Silencing of PARP2
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Antibody | Company | Catalog Number | Dilution |
---|---|---|---|
TOMM20 | Abcam (Cambridge, MA, USA) | ab56783 | 1:200 |
LC3A/B Alexa Fluor 488 conjugate | Cell Signaling Technology (Danvers, MA, USA) | 13082 | 1:50 |
Goat-anti mouse IgG Alexa Fluor 546 | Thermo Fisher Scientific (Walthan, MA, USA) | A-11003 | 1:1000 |
Gene Name | Primers |
---|---|
COX17 | 5′-CGTGATGCGTGCATCATTGA-3′ 5′-CATTCACAAAGTAGGCCACC-3′ |
Cyclophilin | 5′-TGGAGAGCACCAAGACAGACA-3′ 5′-TGCCGGAGTCGACAATGAT-3′ |
Cyt C | 5′-TCCATCAGGGTATCCTCTCC-3′ 5′-GGAGGCAAGCATAAGACTGG-3′ |
Drp1 | 5′-TCGAGTCCCCATTCATTGCAGT-3′ 5′-GAAGAAGGTCCCTGCCCACTAG-3′ |
Fis1 | 5′-CTGGACTCATTGGACTGGCTGT-3′ 5′-AGAGGTAGACTACAGGGGTGCA-3′ |
Mfn1 | 5′-GGTCCTGCAATCACTCTGTCCT-3′ 5′-CCCATTTCACCCCTTCAGACCT-3′ |
Mfn2 | 5′-GTGATCAGGTTCAGCGTCCTCT-3′ 5′-CCACTCCTCCGACCACAAGAAT-3′ |
Ndufa2 | 5′-GCACACATTTCCCCACACTG-3′ 5′-CCCAACCTGCCCATTCTGAT-3′ |
Ndufb3 | 5′-TACCACAAACGCAGCAAACC-3′ 5′-AAGGGACGCCATTAGAAACG-3′ |
Ndufb5 | 5′-CTTCGAACTTCCTGCTCCTT-3′ 5′-GGCCCTGAAAAGAACTACG-3′ |
Nox4 | 5′-GCAGATTTACTCTGTGTGTTGCAT-3′ 5′-TCCCATCTGTTTGACTGAGGT-3′ |
Opa1 | 5′-ATTGTGTGCTCTCAGTCAGGCT-3′ 5′-ACCTTTCCCTGACGCCTAGTTC-3′ |
Parkin | 5′-AAATGCATCTGGAGGGGACGAA-3′ 5′-TAACTGGACCTCTGGCTGCTTC-3′ |
Parp1 | 5′-GGAGCTGCTCATCTTCAACC-3′ 5′-GCAGTGACATCCCCAGTACA-3′ |
Parp2 | 5′-GGAAGGCGAGTGCTAAATGAA-3′ 5′-AAGGTCTTCACAGAGTCTCGATTG-3′ |
Parp3 | 5′-CCTGCTGATAATCGGGTCAT-3′ 5′-TTGTTGTTGTTGCCGATGTT-3′ |
Pink1 | 5′-GGGAAGAACAGCCTTGAACAGC-3′ 5′-GCAAAGTTCAGTGTTGGCCTCA-3′ |
Sirt1 | 5′-TGTGAAGTTACTGCAGGAGTGTAAA-3′ 5′-GCATAGATACCGTCTCTTGATCTGAA-3′ |
36B4 | 5′-AGATTCGGGATATGCTGTTGG-3′ 5′-AAAGCCTGGAAGAAGGAGGTC-3′ |
Antibody | Company | Catalog Number | Dilution |
---|---|---|---|
GAPDH | Sigma-Aldrich | G9545 | 1:10,000 |
HSP25 | Enzo Life Sciences | ADI-SPA-801 | 1:1500 |
HSP70/HSP72 (C92F3A-5) | Enzo Life Sciences | ADI-SPA-810 | 1:8000 |
HSC70/HSP70 (N27F3-4) | Enzo Life Sciences | ADI-SPA-820 | 1:1000 |
HSP40 (C-20) | Santa Cruz Biotechnology | sc-1800 | 1:1000 |
HSP90 (AC88) | Enzo Life Sciences | ADI-SPA-830 | 1:1000 |
HSP110 | Becton Dickinson Biosciences | 610510 | 1:2500 |
iNOS | Novus Biologicals | NB300-605 | 1:1000 |
NTyr | Millipore | 06-284 | 1:1000 |
OPA1 | Thermo Fisher Scientific | MA5-16149 | 1:1000 |
Parkin | Thermo Fisher Scientific | PA5-13399 | 1:1000 |
PARP1 | Cell Signaling Technology | 9532 | 1:2000 |
PARP2 | Enzo Life Sciences | ALX-210-899 | 1:2000 |
PARP3 | Novus Biologicals | NBP1-31415 | 1:1000 |
PAR | Sigma-Aldrich | MABC547 | 1:1000 |
PINK1 | Novus Biologicals | BC100-494 | 1:1000 |
SIRT1 | Millipore | 07-131 | 1:1000 |
4HNE | Abcam | ab46545 | 1:1000 |
Anti-mouse IgG, HRP-linked | Sigma-Aldrich | A9044 | 1:2000 |
Anti-rabbit IgG, HRP-linked | Cell Signaling Technology | 7074 | 1:2000 |
Anti-β-actin-peroxidase | Sigma-Aldrich | A3854 | 1:20,000 |
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Jankó, L.; Kovács, T.; Laczik, M.; Sári, Z.; Ujlaki, G.; Kis, G.; Horváth, I.; Antal, M.; Vígh, L.; Bálint, B.L.; et al. Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation. Cells 2021, 10, 1387. https://doi.org/10.3390/cells10061387
Jankó L, Kovács T, Laczik M, Sári Z, Ujlaki G, Kis G, Horváth I, Antal M, Vígh L, Bálint BL, et al. Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation. Cells. 2021; 10(6):1387. https://doi.org/10.3390/cells10061387
Chicago/Turabian StyleJankó, Laura, Tünde Kovács, Miklós Laczik, Zsanett Sári, Gyula Ujlaki, Gréta Kis, Ibolya Horváth, Miklós Antal, László Vígh, Bálint L. Bálint, and et al. 2021. "Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation" Cells 10, no. 6: 1387. https://doi.org/10.3390/cells10061387
APA StyleJankó, L., Kovács, T., Laczik, M., Sári, Z., Ujlaki, G., Kis, G., Horváth, I., Antal, M., Vígh, L., Bálint, B. L., Uray, K., & Bai, P. (2021). Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation. Cells, 10(6), 1387. https://doi.org/10.3390/cells10061387