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Updated: Apr 2, 2026
Cellular Redox Profiling Using High-content Microscopy
Published on: May 14, 2017
Cross Talk in HEK293 Cells Between Nrf2, HIF, and NF-κB Activities upon Challenges with Redox Therapeutics
Katarina Johansson1, Marcus Cebula1, Olle Rengby1
11 Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Stockholm, Sweden .
Aim:
Many transcription factors with importance in health and disease are redox regulated. However, how their activities may be intertwined in responses to redox-perturbing stimuli is poorly understood. To enable in-depth characterization of this aspect, we here developed a methodology for simultaneous determination of nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation at single-cell resolution, using a new tool named pTRAF (plasmid for transcription factor reporter activation based upon fluorescence). The pTRAF allowed determination of Nrf2, HIF, and NF-κB activities in a high-resolution and high-throughput manner, and we here assessed how redox therapeutics affected the activities of these transcription factors in human embryonic kidney cells (HEK293).
Results:
Cross talk was detected between the three signaling pathways upon some types of redox therapeutics, also by using inducers typically considered specific for Nrf2, such as sulforaphane or auranofin, hypoxia for HIF activation, or tumor necrosis factor alpha (TNFα) for NF-κB stimulation. Doxorubicin, at low nontoxic doses, potentiated TNFα-induced activation of NF-κB and HIF, without effects in stand-alone treatment. Stochastic activation patterns in cell cultures were also considerable upon challenges with several redox stimuli.
Innovation:
A novel strategy was here used to study simultaneous activation of Nrf2, HIF, and NF-κB in single cells. The method can also be adapted for studies of other transcription factors.
Conclusion:
The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-κB are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells. Antioxid. Redox Signal. 26, 229-246.
Insights
Researchers developed a new method to simultaneously measure key transcription factors nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB). This revealed complex cross-talk between these pathways in response to redox stimuli.
Area of Science:
- Cellular and Molecular Biology
- Redox Biology
- Signaling Pathways
Background:
- Transcription factors like Nrf2, HIF, and NF-κB are crucial in health and disease.
- Their intricate regulation by redox stimuli and potential cross-talk remain poorly understood.
Purpose of the Study:
- To develop a novel methodology for simultaneous, single-cell resolution measurement of Nrf2, HIF, and NF-κB activation.
- To investigate the impact of redox therapeutics on these transcription factors in HEK293 cells.
Main Methods:
- Development of a new tool, pTRAF (plasmid for transcription factor reporter activation based upon fluorescence).
- High-resolution, high-throughput determination of Nrf2, HIF, and NF-κB activities.
- Assessment of redox therapeutic effects on transcription factor activation.
Main Results:
- Detected cross-talk between Nrf2, HIF, and NF-κB signaling pathways under specific redox conditions.
- Observed potentiated NF-κB and HIF activation by low-dose doxorubicin in combination with TNFα.
- Identified stochastic activation patterns in response to various redox stimuli.
Conclusions:
- The pTRAF method enables in-depth studies of transcription factor dynamics.
- Nrf2, HIF, and NF-κB show unique responses to stimuli but also exhibit significant cross-talk within single cells.
- This provides new insights into cellular responses to redox perturbations.
