Blue Light Damage and p53: Unravelling the Role of p53 in Oxidative-Stress-Induced Retinal Apoptosis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Organ Cultures
Isolation and Cultivation of Retinal Explants
2.2. Light Sources and Exposure Set Ups
Blue and White Light Exposure
2.3. Methods
2.3.1. Ex Vivo Optical Coherence Tomography (OCT)
2.3.2. Quantification of the Retinal Sections (OCT Measurements)
2.3.3. Cell Viability, ROS Assay, and Caspase 3/7 Activity Assay
2.3.4. Immunostainings
2.3.5. Western Blot
2.3.6. Inhibition of p53
2.3.7. Quantitative Real-Time PCR
2.3.8. TUNEL (TdT-Mediated dUTP-Biotin Nick end Labeling) Analysis
2.3.9. Statistical Analysis
2.3.10. Image Generation
3. Results
3.1. BL Is the Damaging Component of WL
3.2. Müller Cells React to BL Exposure
3.3. BL Damages Retinal Cells and Leads to Accumulation of Rhodopsin
3.4. Apoptosis Induced by Blue Light Depends on the Exposure Duration
3.5. BL Induced the Expression of Modified p53
3.6. BL-Induced Retinal Damage Can Be Reduced by p53 Inhibition
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Antibody | Company Primary AB | Dilution Primary AB | Company Secondary AB | Dilution Secondary AB |
---|---|---|---|---|
acetylated (Lys382) p53 | Cell Signaling (Danvers, MA, USA) | 1:100 | Thermo Fisher Scientific (Invitrogen; Karlsruhe, Germany) | 1:1000 Alexa Fluor 488 |
GFAP | BD Bioscience (BP Pharmigen, Heidelberg, Germany) | 1:200 | Thermo Fisher Scientific (Karlsruhe, Germany) | 1:1000 Alexa Fluor 555 |
Rhodopsin | Abcam (Cambridge, UK) | 1:200 | Thermo Fisher Scientific (Invitrogen; Karlsruhe, Germany) | 1:1000 Alexa Fluor 594 |
Primary Antibody | ||
---|---|---|
Target | Company | Dilution |
β-Actin | Cell Signaling, Danvers, MA, USA | 1:1000 |
HSP70 | Santa Cruz, Dallas, TX, USA | 1:500 |
NF-kB | Elabscience, Hamburg, Germany | 1:500 |
Rhodopsin | Abcam, Cambridge, UK | 1:200 |
BAX | Cell Signaling, Danvers, MA, USA | 1:100 |
BCL2 | Cell Signaling, Danvers, MA, USA | 1:100 |
Secondary Antibody | ||
Target | Company | Dilution |
mouse | IRDye® 680RD, LI-COR, Homburg, Germany | 1:10,000 |
rabbit | IRDye® 800CW, LI-COR, Homburg, Germany | 1:10,000 |
Gene | Sequence 5′-3′ | |
---|---|---|
Actin-β for | CACGCCATCCTGCGTCTGGA | XM_003357928.4 |
Actin-β rev | AGCACCGTGTTGGCGTAGAG | |
RPL4 for | CAAGAGTAACTACAACCTTC | XM_005659862.3 |
RPL4 rev | GAACTCTACGATGAATCTTC | |
GFAP for | GGAGAAGCCTTTGCTACACG | NM_001244397.1 |
GFAP rev | TCTTCACTCTGCCTGGGTCT | |
Opsin (mws) for | GGGGAGCATCTTCACCTACA | NM_001011506.1 |
Opsin (mws) rev | GATGATGGTCTCTGCCAGGT | |
Rhodopsin for | TCCAGGTACATCCCAGAAGG | NM_214221.1 |
Rhodopsin rev | GCTGCCCATAGCAGAAGAAG | |
β-III-tubulin for | CAGATGTTCGATGCCAAGAA | AK391872.1 |
β-III-tubulin rev | GGGATCCACTCCACGAAGTA | |
TNF-α for | CCACCAACGTTTTCCTCACT | JF831365.1 |
TNF-α rev | CCAAAATAGACCTGCCCAGA | |
PKCa for | ACCGAACAACAAGGAACGAC | XM_021066740.1 |
PKCa rev | CTGAGCTCCACGTTTCCTTC | |
HSP70 for | ATGTCCGCTGCAAGAGAAGT | NM_001123127.1 |
HSP70 rev | GGCGTCAAACACGGTATTCT | |
NF-kB for | AGGATGGGATCTGCACTGTC | NM_001048232.1 |
NF-kB rev | ATCAGGGTGCACCAAAAGTC | |
BAX for | AAGCGCATTGGAGATGAACT | XM_003127290.5 |
BAX rev | AAAGTAGAAAAGCGCGACCA | |
Bcl-2 for | AATTACCATCGGCGTAGTGC | XM_021099593.1 |
Bcl-2 rev | CGTTTCAGCCACCGTAAAAT |
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Fietz, A.; Corsi, F.; Hurst, J.; Schnichels, S. Blue Light Damage and p53: Unravelling the Role of p53 in Oxidative-Stress-Induced Retinal Apoptosis. Antioxidants 2023, 12, 2072. https://doi.org/10.3390/antiox12122072
Fietz A, Corsi F, Hurst J, Schnichels S. Blue Light Damage and p53: Unravelling the Role of p53 in Oxidative-Stress-Induced Retinal Apoptosis. Antioxidants. 2023; 12(12):2072. https://doi.org/10.3390/antiox12122072
Chicago/Turabian StyleFietz, Agnes, Francesca Corsi, José Hurst, and Sven Schnichels. 2023. "Blue Light Damage and p53: Unravelling the Role of p53 in Oxidative-Stress-Induced Retinal Apoptosis" Antioxidants 12, no. 12: 2072. https://doi.org/10.3390/antiox12122072
APA StyleFietz, A., Corsi, F., Hurst, J., & Schnichels, S. (2023). Blue Light Damage and p53: Unravelling the Role of p53 in Oxidative-Stress-Induced Retinal Apoptosis. Antioxidants, 12(12), 2072. https://doi.org/10.3390/antiox12122072