Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase–Luciferin Pair
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Plasmids and Cloning
2.3. Cell Lines, Cell Culture and Transfection
2.4. Protein Sample Preparation and Immunoblotting
2.5. Quantitative RT-PCR
2.6. Live Cell Imaging
2.7. Bioluminescence Imaging (BLI) of Tumor Xenografts
2.8. Chromatin Immunoprecipitation
2.9. Statistical Analysis
3. Results
3.1. Human Codon Optimization Improved Wild-Type Mushroom Luciferase (Luz) Reporter Activity
3.2. Luz and 3OH-Hispidin Combination Is a Novel Non-Cross-Reacting Bioluminescence Reporter System
3.3. NF-κB Regulatory Response Can Be Measured by Using hLuz Reporter Sensor
3.4. Application of Triple Luciferase Assay Revealed Complex Regulation of PIK3CA Gene Promoter by p53 and NF-κB
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Codon Adaptive Index | GC Content | Codon Frequency Distribution | |
---|---|---|---|
Standard | 0.8–1.0 | 30–70% | <30% |
Luz | 0.71 | 52.15% | 3% |
hLuz | 0.92 | 56.85% | 0% |
Nucleotide sequence alignment of Luz and hLuz | ||||
---|---|---|---|---|
Max score | Total score | Query cover | E value | Identity |
627 | 627 | 96% | 0.0 | 78% |
Amino acid sequence alignment of Luz and hLuz | ||||
Max score | Total score | Query cover | E value | Identity |
554 | 554 | 100% | 0.0 | 100% |
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Mujawar, A.; Phadte, P.; Palkina, K.A.; Markina, N.M.; Mohammad, A.; Thakur, B.L.; Sarkisyan, K.S.; Balakireva, A.V.; Ray, P.; Yampolsky, I.; et al. Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase–Luciferin Pair. Sensors 2023, 23, 7313. https://doi.org/10.3390/s23177313
Mujawar A, Phadte P, Palkina KA, Markina NM, Mohammad A, Thakur BL, Sarkisyan KS, Balakireva AV, Ray P, Yampolsky I, et al. Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase–Luciferin Pair. Sensors. 2023; 23(17):7313. https://doi.org/10.3390/s23177313
Chicago/Turabian StyleMujawar, Aaiyas, Pratham Phadte, Ksenia A. Palkina, Nadezhda M. Markina, Ameena Mohammad, Bhushan L. Thakur, Karen S. Sarkisyan, Anastasia V. Balakireva, Pritha Ray, Ilia Yampolsky, and et al. 2023. "Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase–Luciferin Pair" Sensors 23, no. 17: 7313. https://doi.org/10.3390/s23177313