Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Models
2.2. Animal Handling and Allograft Tumour Model
2.3. Transmission Electron Microscopy
2.4. Immunofluorescence and Mitochondria Morphometric Analysis
2.5. Quantitative Real Time-PCR (qPCR)
2.6. Protein Isolation and Western Blotting
2.7. Mitochondria Respiratory Rate
2.8. ATP Production
2.9. Mitochondrial Membrane Potential Analysis
2.10. Statistical Analysis
3. Results
3.1. A-SMase Expression Determines Mitochondrial Morphology
3.2. A-SMase Expression Regulates Mitochondrial Elongation through Mfn1 and OPA1
3.3. A-SMase Downregulation Improves Mitochondrial Function
3.4. A-SMase Downregulation Increases Mitochondrial Biogenesis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene Accession Number | Primer Sequence | Amplicon | |
---|---|---|---|
A-SMase (smpd1) | NM_011421 | F: 5′-TGGGACTCCTTTGGATGGG-3′ R: 5′-CGGCGCTATGGCACTGAAT-3′ | 134 bp |
Mfn1 | NM_024200 | F: 5′-CCTACTGCTCCTTCTAACCCA-3′ R: 5′-AGGGACGCCAATCCTGTGA-3′ | 86 bp |
Mfn2 | NM_133201 | F: 5′-AGAACTGGACCCGGTTACCA-3′ R: 5′-CACTTCGCTGATACCCCTGA-3′ | 82 bp |
OPA1 | NM_133752 | F: 5′-TGGAAAATGGTTCGAGAGTCAG-3′ R: 5′-CATTCCGTCTCTAGGTTAAAGCG-3′ | 76 bp |
Drp1 | NM_152816 | F: 5′-GCTGGATCACGGGACAAGTTAA-3′ R: 5′-TGCCTGTTGTTGGTTCCTGAC-3′ | 106 bp |
Mitf | NM_001113198 NM_008601 NM_001178049 | F: 5′-CCAACAGCCCTATGGCTATGC-3′ R: 5′-CTGGGCACTCACTCTCTGC-3′ | 99 bp |
HK2 | NM_013820 | F: 5′-TGATCGCCTGCTTATTCACGG-3′ R: 5′-AACCGCCTAGAAATCTCCAGA-3′ | 112 bp |
PFKFB3 | NM_001177752 | F: 5′-CCCAGAGCCGGGTACAGAA-3′ R: 5′-GGGGAGTTGGTCAGCTTCG-3′ | 88 bp |
CS | NM_026444 | F: 5′-GGACAATTTTCCAACCAATCTGC-3′ R: 5′-TCGGTTCATTCCCTCTGCATA-3′ | 109 bp |
MDH | NM_008617 | F: 5′-TTGGGCAACCCCTTTCACTC-3′ R: 5′-GCCTTTCACATTTGCTCTGGTC-3′ | 131 bp |
IDH2 | NM_173011 | F: 5′-GGAGAAGCCGGTAGTGGAGAT-3′ R: 5′-GGTCTGGTCACGGTTTGGAA-3′ | 139 bp |
IDH3G | NM_008323 | F: 5′-GGTGCTGCAAAGGCAATGC-3′ R: 5′-TATGCCGCCCACCATACTTAG-3′ | 136 bp |
SDHA | NM_023281 | F: 5′-GGAACACTCCAAAAACAGACCT-3′ R: 5′-CCACCACTGGGTATTGAGTAGAA-3′ | 106 bp |
COX I | NC_005089.1 | F: 5′-CCAGTGCTAGCCGCAGGCAT-3′ R: 5′-GCTGGTAGAGAATTGGGTCCCCTCC-3′ | 100 bp |
COX IV | NM_009941 | F: 5′-TACTTCGGTGTGCCTTCGA-3′ R: 5′-TTAGCATGGACCATTGGATACGG-3′ | 110 bp |
CYT B | NC_005089.1 | F: 5′-ACGCCATTCTACGCTCAATC -3′ R: 5′-GCTTCGTTGCTTTGAGGTAT-3′ | 110 bp |
CYT C | NM_007808 | F: 5′-ATAGGGGCATGTCACCTCAAAC-3′ R: 5′-GTGGTTAGCCATGACCTGAAAG-3′ | 172 bp |
ATPase | NM_016774 | F: 5′-CGTGAGGGCAATGATTTATACCAT-3′ R: 5′-TCCTGGTCTCTGAAGTATTCAGCAA-3′ | 170 bp |
mtDNA | NC_005089 | F: 5′-CCTATCACCCTTGCCATCAT-3′ R: 5′-GAGGCTGTTGCTTGTGTGAC-3′ | 194 bp |
RNase P (DNA) | NC_000085 | F: 5′-GAAGGCTCTGCGCGGACTCG-3′ R: 5′-CGAGAGACCGGAATGGGGCCT-3′ | 119 bp |
PGC-1alpha | NM_008904 | F: 5′-ACTATGAATCAAGCCACTACAGAC-3′ R: 5′-TTCATCCCTCTTGAGCCTTTCG-3′ | 143 bp |
TFAM | NM_009360 | F: 5′-AAGACCTCGTTCAGCATATAACATT-3′ R: 5′-TTTTCCAAGCCTCATTTACAAGC-3′ | 104 bp |
36b4 | NM_007475 | F: 5′-AGGATATGGGATTCGGTCTCTTC-3′ R: 5′-TCATCCTGCTTAAGTGAACAAACT-3′ | 143 bp |
RPL32 | NM_172086 | F: 5′-TTAAGCGAAACTGGCGGAAAC-3′ R: 5′-TTGTTGCTCCCATAACCGATG-3′ | 100 bp |
Actin beta | NM_007393 | F: 5′-GGCTGTATTCCCCTCCATCG-3′ R: 5′-CCAGTTGGTAACAATGCCATGT-3′ | 154 bp |
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Coazzoli, M.; Napoli, A.; Roux-Biejat, P.; De Palma, C.; Moscheni, C.; Catalani, E.; Zecchini, S.; Conte, V.; Giovarelli, M.; Caccia, S.; et al. Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma. Cells 2020, 9, 848. https://doi.org/10.3390/cells9040848
Coazzoli M, Napoli A, Roux-Biejat P, De Palma C, Moscheni C, Catalani E, Zecchini S, Conte V, Giovarelli M, Caccia S, et al. Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma. Cells. 2020; 9(4):848. https://doi.org/10.3390/cells9040848
Chicago/Turabian StyleCoazzoli, Marco, Alessandra Napoli, Paulina Roux-Biejat, Clara De Palma, Claudia Moscheni, Elisabetta Catalani, Silvia Zecchini, Vincenzo Conte, Matteo Giovarelli, Sonia Caccia, and et al. 2020. "Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma" Cells 9, no. 4: 848. https://doi.org/10.3390/cells9040848
APA StyleCoazzoli, M., Napoli, A., Roux-Biejat, P., De Palma, C., Moscheni, C., Catalani, E., Zecchini, S., Conte, V., Giovarelli, M., Caccia, S., Procacci, P., Cervia, D., Clementi, E., & Perrotta, C. (2020). Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma. Cells, 9(4), 848. https://doi.org/10.3390/cells9040848