Monitoring and Regulating Intracellular GPX4 mRNA Using Gold Nanoflare Probes and Enhancing Erastin-Induced Ferroptosis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Instrumentation
2.3. Experimental Sections
2.3.1. Preparation of Gold Nanoparticles and Gold Nanoflare Probes
2.3.2. Incubation of AuNF Probes with Target-DNA or Cell Lysis Solution
2.3.3. Cell Culture, Confocal Imaging, and Flow Cytometric Analysis
2.3.4. qRT-PCR Analysis
2.3.5. Immunofluorescence Labeling
2.3.6. Determining the Content of Glutathione Peroxidases
2.3.7. Inhibiting the Proliferation of Cancer Cells through Erastin or the Synergistic Effect of Erastin and AuNF Probes
2.3.8. Determining the Content Change of Intracellular ROS Caused by Erastin and AuNF Probes
2.3.9. Statistical Analysis
3. Results and Discussion
3.1. Design of AuNF Probes and Mechanism of Enhancing Erastin-Induced Ferroptosis
3.2. Characterization of AuNPs and AuNF Probes
3.3. Fluorescence Response of AuNF Probes to Target-DNA and Cell Lysis Solution
3.4. Evaluation the Number of HS-DNA/Flare-DNA Duplexes Bound on AuNF Probes
3.5. Fluorescence Imaging of Intracellular GPX4 mRNA by CLSM
3.6. Expression Analysis of Intracellular GPX4 mRNA, GPX4 and Total GPX
3.7. Enhancing Erastin-Induced Ferroptosis Using the Synergistic Effect of AuNF Probes
3.8. ROS Generation Caused by AuNF Probes and Erastin
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Names | Sequences |
---|---|
HS-DNA | 5′-TCA GCG TAT CAA AAA AAA AA-SH-3′ |
HS-DNA’ | 5′-GAT CTA AGC TAA AAA AAA AA-SH-3′ |
Flare-DNA | 5′-GAT ACG CTG AGT GTG GTT T-Cy5-3′ |
Flare-DNA’ | 5′-AGC TTA GAT CGC TAT GTA C-Cy5-3′ |
Flare-DNA-N | 5′-GAT ACG CTG AGT GTG GTT T-3′ |
Target-DNA | 5′-AAA CCA CAC TCA GCG TAT C-3′ |
Random-DNA | 5′-GGC CGA TTG TGA ACA TGG A -3′ |
GPX4-F-primer | 5′-AGA GAT CAA AGA GTT CGC CGC-3′ |
GPX4-R-primer | 5′-TCT TCA TCC ACT TCC ACA GCG-3′ |
GAPDH-F-primer | 5′-CTC AGA CAC CAT GGG GAA GGT GA-3′ |
GAPDH-R-primer | 5′-ATG ATC TTG AGG CTG TTG TCA TA-3′ |
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Liu, X.; Yang, Q.; Sui, Y.; Yue, Q.; Yan, S.; Li, C.; Hong, M. Monitoring and Regulating Intracellular GPX4 mRNA Using Gold Nanoflare Probes and Enhancing Erastin-Induced Ferroptosis. Biosensors 2022, 12, 1178. https://doi.org/10.3390/bios12121178
Liu X, Yang Q, Sui Y, Yue Q, Yan S, Li C, Hong M. Monitoring and Regulating Intracellular GPX4 mRNA Using Gold Nanoflare Probes and Enhancing Erastin-Induced Ferroptosis. Biosensors. 2022; 12(12):1178. https://doi.org/10.3390/bios12121178
Chicago/Turabian StyleLiu, Xiaoyan, Qiangqiang Yang, Yanan Sui, Qiaoli Yue, Shuqing Yan, Chuan Li, and Min Hong. 2022. "Monitoring and Regulating Intracellular GPX4 mRNA Using Gold Nanoflare Probes and Enhancing Erastin-Induced Ferroptosis" Biosensors 12, no. 12: 1178. https://doi.org/10.3390/bios12121178