Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide †
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and Treatment
2.2. RNA Isolation and Real-Time Quantitative RT-PCR
2.3. Western Blot Analysis
2.4. Detection of Mitochondrial Superoxide with MitoSOX
2.5. Measurement of Cellular Respiration
2.6. Measurement of Cellular Glycolysis
2.7. Cell Migration Assay
2.8. Dispase-Induced PVR in Mouse
2.9. Immunofluorescence
2.10. Enzyme-Linked Immunosorbent Assay (ELISA)
2.11. Statistical Analysis
3. Results
3.1. αBC-P Suppressed EMT-Associated Genes in hRPE Cells
3.2. αBC-P Suppressed EMT-Associated Proteins in hRPE Cells
3.3. αBC-ELP Treatment Inhibited Mitochondrial ROS Production in hRPE Cells Exposed to TGFβ2
3.4. αBC-ELP Treatment Inhibited SMAD4 Mitochondrial Translocation in hRPE Cells
3.5. TGFβ2-Induced Changes in Energy Metabolism and the Role of αBC-P in RPE
3.6. αBC-P Inhibits TGFβ2-Induced Migration of hRPE Cells
3.7. αBC-P Inhibits Matrix Deposition in Dispase-Induced PVR in Mice
3.8. Evidence for αBC-ELP-Induced Improvement in RPE Cell Function by ERG
3.9. αBC-ELP Inhibits EMT in Dispase-Induced PVR in Mice
3.10. αBC-ELP Suppressed EMT/MET-Associated Proteins in Dispase-Induced PVR
3.11. αBC-ELP Suppressed CTGF in Dispase-Induced PVR in Mice
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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Target Molecule | Accession Number | Forward Primer Sequence | Reverse Primer Sequence |
---|---|---|---|
αSMA | NM_001613 | 5′-TCTGTAAGGCCGGCTTTGC-3′ | 5′-TGTCCCATTCCCACCATCA-3′ |
Vimentin | NM_003380 | 5′-TGAGTACCGGAGACAGGTGCAG-3′ | 5′-TAGCAGCTTCAACGGCAAAGTTC-3′ |
E-cadherin | NM_004360 | 5′-ATTTTTCCCTCACACCCGAT-3′ | 5′-TCCCAGGCGTAGACCAAGA-3′ |
Sirt1 | NM_012238 | 5′-TCCTGGACAATTCCAGCCATCTCT-3′ | 5′-TTCCAGCGTTATGTTCTGGGT-3′ |
GAPDH | NM_002046 | 5′-ACAGTCGCCGCATCTTCTT-3′ | 5′-CTTGATTTTGGAGGGATCTCGC-3′ |
Target Molecule | Antibody Type | Source | Dilution |
---|---|---|---|
αSMA (ab7817) | Mouse monoclonal | Abcam | 1:1000 |
E-cadherin (24E10) | Rabbit monoclonal | Cell Signaling | 1:1000 |
pSMAD2/3 ((S465/467)/(S423/425) | Rabbit monoclonal | Cell Signaling | 1:1000 |
SMAD2/3 (D7G7) | Rabbit monoclonal | Cell Signaling | 1:1000 |
Fibronectin (EP5) | Mouse monoclonal | Santa Cruz Biotechnology | 1:1000 |
Vimentin (3CB2) | Mouse monoclonal | Santa Cruz Biotechnology | 1:1000 |
SNAIL (C15D3) | Rabbit monoclonal | Cell Signaling | 1:1000 |
SLUG (G-18) | Goat polyclonal | Santa Cruz Biotechnology | 1:1000 |
CTGF (ab6992) | Rabbit monoclonal | Abcam | 1:1000 |
GAPDH (MAB374) | Mouse monoclonal | EMD Millipore | 1:1000 |
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Wada, I.; Sreekumar, P.G.; Spee, C.; MacKay, A.J.; Ip, M.; Kannan, R. Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide. Antioxidants 2022, 11, 2080. https://doi.org/10.3390/antiox11102080
Wada I, Sreekumar PG, Spee C, MacKay AJ, Ip M, Kannan R. Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide. Antioxidants. 2022; 11(10):2080. https://doi.org/10.3390/antiox11102080
Chicago/Turabian StyleWada, Iori, Parameswaran G Sreekumar, Christine Spee, Andrew J MacKay, Michael Ip, and Ram Kannan. 2022. "Mechanisms of Epithelial-Mesenchymal Transition and Prevention of Dispase-Induced PVR by Delivery of an Antioxidant αB Crystallin Peptide" Antioxidants 11, no. 10: 2080. https://doi.org/10.3390/antiox11102080