In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream
Abstract
1. Introduction
2. Materials and Methods
2.1. Antibodies used for Western Blot
2.2. Cell Lines and Culture Conditions
2.3. Densitometric Analysis
2.4. Quantitative RT-PCR
3. Results
3.1. The Response of Hypoxia Response Proteins to Hypoxia and Reoxygenation
3.2. Cellular Responses to Hypoxia and Reoxygenation in Breast Cancer-Relevant Proteins
3.3. Changes of Epithelial Differentiation Marker Proteins under Hypoxia and Reoxygenation
3.4. Cellular Responses to Hypoxia and Reoxygenation on mRNA Level
3.5. Discordance of mRNA and Protein Levels under Standard Conditions, Hypoxia, and Reoxygenation
3.6. Hypoxia and Reoxygenation Affected the Protein Phosphorylation Status
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Gene Product | Cell Line | ΔCq Under S (mRNA) | Condition | x-Fold Change (mRNA) | STDEV (mRNA) | x-Fold Change (Protein) | STDEV (Protein) | p-Value Protein Level vs. mRNA Level | p-Value Hyp vs. Reox (Protein Level) | p-Value Hyp vs. Reox (mRNA Level) |
---|---|---|---|---|---|---|---|---|---|---|
PD-L1 | MDA-MB-231 | 6.73 | Hyp | 0.56 | 0.06 | 1.98 | 0.44 | 0.029 | 0.004 | 0.045 |
Reox | 1.23 | 0.09 | 1.57 | 0.40 | 0.277 | |||||
MDA-MB-468 | 13.97 | Hyp | 0.66 | 0.27 | no signals | NA | NA | 0.347 | ||
Reox | 0.47 | 0.08 | no signals | NA | ||||||
MCF-7 | 16.52 | Hyp | 1.70 | 0.40 | no signals | NA | NA | 0.036 | ||
Reox | 0.69 | 0.15 | no signals | NA | ||||||
BC-M1 | 6.52 | Hyp | 1.07 | 0.06 | 3.65 | 0.69 | 0.022 | 0.024 | 0.001 | |
Reox | 0.68 | 0.06 | 2.00 | 0.66 | 0.073 | |||||
Vimentin | MDA-MB-231 | −2.78 | Hyp | 0.94 | 0.10 | 0.95 | 0.02 | 0.880 | 0.422 | 0.496 |
Reox | 0.89 | 0.05 | 0.98 | 0.06 | 0.119 | |||||
MDA-MB-468 | 7.11 | Hyp | 0.90 | 0.10 | 1.00 1 | 0 | NA | 0.023 | 0.309 | |
Reox | 1.00 | 0.11 | 0.72 | 0.08 | NA | |||||
MCF-7 | 8.26 | Hyp | 0.81 | 0.09 | no signals | NA | NA | 0.178 | ||
Reox | 0.93 | 0.09 | no signals | NA | ||||||
BC-M1 | −3.30 | Hyp | 1.08 | 0.24 | 1.02 | 0.08 | 0.715 | 0.237 | 0.309 | |
Reox | 1.27 | 0.12 | 1.20 | 0.18 | 0.609 | |||||
EGFR | MDA-MB-231 | 2.69 | Hyp | 0.87 | 0.15 | 1.90 | 0.41 | 0.037 | 0.028 | 0.006 |
Reox | 1.50 | 0.13 | 1.11 | 0.35 | 0.185 | |||||
MDA-MB-468 | 0.61 | Hyp | 1.17 | 0.14 | 3.05 | 0.79 | 0.050 | 0.887 | 0.572 | |
Reox | 1.23 | 0.09 | 3.09 | 0.50 | 0.021 | |||||
MCF-7 | 8.40 | Hyp | 1.10 | 0.16 | 21.58 | 16.50 | 0.165 | 0.227 | 0.496 | |
Reox | 1.02 | 0.08 | 49.76 | 11.89 | 0.019 | |||||
BC-M1 | 3.51 | Hyp | 1.17 | 0.10 | 4.99 | 0.38 | 0.002 | 0.133 | 0.002 | |
Reox | 0.53 | 0.05 | 3.50 | 1.39 | 0.066 | |||||
EpCAM | MDA-MB-231 | 4.33 | Hyp | 1.04 | 0.19 | no signals | NA | NA | 0.366 | |
Reox | 0.91 | 0.09 | no signals | NA | ||||||
MDA-MB-468 | −0.21 | Hyp | 0.71 | 0.08 | 0.43 | 0.05 | 0.011 | 0.178 | 0.010 | |
Reox | 1.01 | 0.08 | 0.34 | 0.11 | 0.002 | |||||
MCF-7 | 0.80 | Hyp | 0.94 | 0.10 | 1.54 | 0.28 | 0.052 | 0.089 | 0.081 | |
Reox | 1.13 | 0.10 | 0.95 | 0.25 | 0.341 | |||||
BC-M1 | 10.65 | Hyp | 0.88 | 0.08 | no signals | NA | NA | 0.022 | ||
Reox | 1.38 | 0.17 | no signals | NA | ||||||
ErbB2 | MDA-MB-231 | 8.91 | Hyp | 0.09 | 0.03 | 1.06 | 0.18 | 0.010 | 0.370 | 0.003 |
Reox | 1.94 | 0.20 | 0.97 | 0.30 | 0.013 | |||||
MDA-MB-468 | 13.42 | Hyp | 23.26 | 2.39 | 6.14 | 0.64 | 0.004 | 0.618 | 0.003 | |
Reox | 1.71 | 0.50 | 6.94 | 2.66 | 0.072 | |||||
MCF-7 | 7.19 | Hyp | 0.89 | 0.09 | 0.86 | 0.19 | 0.822 | 0.199 | 0.622 | |
Reox | 0.96 | 0.20 | 0.97 | 0.29 | 0.963 | |||||
BC-M1 | 6.44 | Hyp | 1.85 | 0.23 | 1.61 | 0.29 | 0.327 | 0.247 | 0.006 | |
Reox | 0.18 | 0.03 | 1.29 | 0.10 | 0.001 | |||||
ERα | MDA-MB-231 | 19.09 | Hyp | 1.71 | 0.45 | no signals | NA | NA | 0.052 | |
Reox | 4.02 | 1.10 | no signals | NA | ||||||
MDA-MB-468 | 14.55 | Hyp | 1.16 | 0.28 | no signals | NA | NA | 0.072 | ||
Reox | 0.65 | 0.22 | no signals | NA | ||||||
MCF-7 | 4.59 | Hyp | 0.91 | 0.07 | 0.23 | 0.01 | 0.003 | 0.072 | 0.027 | |
Reox | 1.25 | 0.13 | 0.33 | 0.05 | 0.003 | |||||
BC-M1 | 13.12 | Hyp | 1.39 | 0.15 | no signals | NA | NA | 0.003 | ||
Reox | 0.52 | 0.08 | no signals | NA | ||||||
Grp78 | MDA-MB-231 | 1.83 | Hyp | 0.43 | 0.04 | 0.86 | 0.04 | <0.001 | 0.672 | 0.003 |
Reox | 1.14 | 0.10 | 0.83 | 0.15 | 0.003 | |||||
MDA-MB-468 | 5.92 | Hyp | 2.90 | 0.27 | 0.49 | 0.14 | 0.001 | 0.085 | 0.003 | |
Reox | 0.85 | 0.08 | 0.87 | 0.13 | 0.834 | |||||
MCF-7 | 3.63 | Hyp | 1.11 | 0.10 | 0.36 | 0.11 | 0.001 | 0.903 | 0.227 | |
Reox | 1.01 | 0.06 | 0.34 | 0.11 | 0.002 | |||||
BC-M1 | 2.40 | Hyp | 1.12 | 0.11 | 1.14 | 0.15 | 0.862 | 0.295 | 0.003 | |
Reox | 0.44 | 0.05 | 1.31 | 0.35 | 0.047 |
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Bartkowiak, K.; Koch, C.; Gärtner, S.; Andreas, A.; Gorges, T.M.; Pantel, K. In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream. Cells 2020, 9, 1316. https://doi.org/10.3390/cells9051316
Bartkowiak K, Koch C, Gärtner S, Andreas A, Gorges TM, Pantel K. In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream. Cells. 2020; 9(5):1316. https://doi.org/10.3390/cells9051316
Chicago/Turabian StyleBartkowiak, Kai, Claudia Koch, Sebastian Gärtner, Antje Andreas, Tobias M Gorges, and Klaus Pantel. 2020. "In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream" Cells 9, no. 5: 1316. https://doi.org/10.3390/cells9051316
APA StyleBartkowiak, K., Koch, C., Gärtner, S., Andreas, A., Gorges, T. M., & Pantel, K. (2020). In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream. Cells, 9(5), 1316. https://doi.org/10.3390/cells9051316