The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress
Abstract
:1. Introduction
2. Results
2.1. Effects of Hyperglycemia on RGCs
2.2. Immunoblot Analysis of RGCs with Hyperglycemia
2.3. Immunofluorescence Analysis of RGCs with Hyperglycemia
2.4. Effects in TGF-β1/2 Knockdown RGCs with Hyperglycemia
2.5. Immunoblot Analysis of Activation of Downstream Substrates in TGF-β1/2 Knockdown RGC-5 Cells Treated with Hyperglycemia
2.6. Effects in RGCs with Hyperglycemia with or w/o rhTGF-β1 Protein (5 ng/mL)
2.7. Immunoblot Analysis in RGCs with Hyperglycemia with or w/o Recombinant TGF-β1 Protein (5 ng/mL)
2.8. Effects in RGCs with Hyperglycemia with or without Hydrogen Peroxide for 1 h
2.9. Immunoblot Analysis in RGCs with Hyperglycemia with or without Hydrogen Peroxide (1 mM) for 1 h
2.10. A Hypothetical Model Detailing the Role of TGF-β in Hyperglycemia
3. Discussion
4. Materials and Methods
4.1. Cell Line and Cell Culture
4.2. Proliferation Assay
4.3. Cell Viability Assay
4.4. Detection of ROS
4.5. Immunoblotting Analysis via Western Blot
4.6. Immunofluorescence
4.7. Treatment of Cells with Recombinant Human TGF-β Protein1 (rhTGF-β1)
4.8. Measurement of Intracellular GSH Content
4.9. High-Resolution Respirometry
4.10. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
References
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Antibody | Molecular Weights (kDa) | Company | Monoclonal/Polyclonal | Working Concentration |
---|---|---|---|---|
Nrf2 | 75/130 | Abclonal A1244 | rab pAb | 1:2000 |
Keap1 | 70 | Abclonal-A17062 | rab pAb | 1:2000 |
HIF-1α | 93 | Abclonal A11945 | rab pAb | 1:2000 |
ALDH3A1 | 50 | Abclonal A13275 | rab pAb | 1:2000 |
HO-1 | 33 | GeneTex-GTX101147 | rab pAb | 1:2000 |
TGFβ-1 | 46 | Abclonal-A2124 | rab pAb | 1:2000 |
TGFβ-2 | 48/55 | Abclonal A3640 | rab pAb | 1:2000 |
LDH (Lactate Dehydrogenase) | 36 | GeneTex-GTX101416 | rab pAb | 1:2000 |
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Chen, H.-Y.; Ho, Y.-J.; Chou, H.-C.; Liao, E.-C.; Tsai, Y.-T.; Wei, Y.-S.; Lin, L.-H.; Lin, M.-W.; Wang, Y.-S.; Ko, M.-L.; et al. The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress. Int. J. Mol. Sci. 2020, 21, 6482. https://doi.org/10.3390/ijms21186482
Chen H-Y, Ho Y-J, Chou H-C, Liao E-C, Tsai Y-T, Wei Y-S, Lin L-H, Lin M-W, Wang Y-S, Ko M-L, et al. The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress. International Journal of Molecular Sciences. 2020; 21(18):6482. https://doi.org/10.3390/ijms21186482
Chicago/Turabian StyleChen, Hsin-Yi, Yi-Jung Ho, Hsiu-Chuan Chou, En-Chi Liao, Yi-Ting Tsai, Yu-Shan Wei, Li-Hsun Lin, Meng-Wei Lin, Yi-Shiuan Wang, Mei-Lan Ko, and et al. 2020. "The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress" International Journal of Molecular Sciences 21, no. 18: 6482. https://doi.org/10.3390/ijms21186482