ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin
Abstract
:1. Introduction
2. Results
2.1. Expression and Purification of Recombinant PcOSM
2.2. Inhibitory Activity of PcOSM on Hyphal Growth of Phytophthora Capsici
2.3. PcOSM Induced Intracellular ROS Accumulation in Leaves
2.4. Molecular Docking and Simulation
2.5. PcOSM Showed no Cytotoxicity in MDA MB231
2.6. ADIPOR1 Localized on Cell Membrane and Showed Co-Localization with PcOSM–ADIPOR1
2.7. PcOSM Induced Disintegration of Cytoskeletal Elements
2.8. PcOSM Increased Intracellular Reactive Oxygen Species (ROS) Levels
2.9. PcOSM Induced Senescence in MDAMB231 Cells
2.10. PcOSM Induced Cell Cycle Arrest in MDAMB231 Cells
3. Discussion
4. Materials and Methods
4.1. Plants and Maintenance
4.2. Cloning and Expression of P. colubrinum Osmotin in E. coli
4.3. IPTG Induction and Purification of Recombinant PcOSM
4.4. SDS PAGE and Western Blot Analysis
4.5. Protein Purification
4.6. Liquid Chromatography Tandem Mass Spectrometry
4.7. LC-MS/MS Data Analysis
4.8. Fungal Growth Inhibition Bioassays
4.8.1. Infiltration Method
4.8.2. Infection Assay and Trypan Blue Staining
4.9. Measurement of ROS Production
4.10. Docking Studies
4.11. MDA MB231Proliferation Assay
4.12. Immunofluorescence Staining
4.13. ROS Detection Assay–DCFDA Fluorescent Microscopy and Flow Cytometry Assay
4.14. Senescence Assay-Beta Galactosidase Detection
4.15. Cell Cycle Analysis
4.16. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Abbreviations
PcOSM | Full length Piper colubrinum Osmotin (693 bop) |
ROS | Reactive oxygen species |
TBHP | Tert-butyl hydroperoxide |
ADIPOR 1 | Adiponectin Receptor-1 |
IPTG | Isopropyl β-d-1-thiogalactopyranoside |
DCFDA | 2′,7′-Dichlorofluorescein Diacetate |
ADIPOQ | Adiponectin |
NtOSM | Nicotiana tabacum Osmotin |
SA-β-gal | Senescence Associated Beta galactosidase |
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Geetha, R.G.; Krishnankutty Nair Chandrika, S.; Saraswathy, G.G.; Nair Sivakumari, A.; Sakuntala, M. ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin. Molecules 2021, 26, 2239. https://doi.org/10.3390/molecules26082239
Geetha RG, Krishnankutty Nair Chandrika S, Saraswathy GG, Nair Sivakumari A, Sakuntala M. ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin. Molecules. 2021; 26(8):2239. https://doi.org/10.3390/molecules26082239
Chicago/Turabian StyleGeetha, Rajeswari Gopal, Sivakumar Krishnankutty Nair Chandrika, Gayathri G. Saraswathy, Asha Nair Sivakumari, and Manjula Sakuntala. 2021. "ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin" Molecules 26, no. 8: 2239. https://doi.org/10.3390/molecules26082239
APA StyleGeetha, R. G., Krishnankutty Nair Chandrika, S., Saraswathy, G. G., Nair Sivakumari, A., & Sakuntala, M. (2021). ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin. Molecules, 26(8), 2239. https://doi.org/10.3390/molecules26082239