Human KEAP1 Knockout Cell Line-HeLa
Cat.No. : CSC-RT0564
Host Cell: HeLa Target Gene: KEAP1
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0564 |
| Cell Line Information | A stable cell line with a homozygous knockout of human KEAP1 using CRISPR/Cas9. |
| Target Gene | KEAP1 |
| Host Cell | HeLa |
| Shipping | 10^6 cells/tube |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene ID | 9817 |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Media Type | Cells were cultured in DMEM supplemented with 10% fetal bovine serum. |
| Growth Properties | Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2. Split at 80-90% confluence, approximately 1:4-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) DMSO |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
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Background
Case Study
Applications
KEAP1, whose full name is Kelch-like ECH-associated protein 1, is a key regulator of the cellular response to oxidative stress. This protein plays a fundamental role in maintaining cellular homeostasis and protecting cells from damage caused by reactive oxygen species (ROS). Under normal conditions, KEAP1 binds to Nrf2 in the cytoplasm, promoting its ubiquitination and subsequent proteasomal degradation. This binding occurs through a complex that includes KEAP1, Nrf2, and the Cullin 3-based E3 ubiquitin ligase complex. By continuously targeting Nrf2 for degradation, KEAP1 ensures that Nrf2 levels remain low under non-stress conditions, thereby preventing the unnecessary activation of antioxidant genes. However, under oxidative stress or electrophilic conditions, KEAP1 undergoes modifications at its cysteine residues, reducing its ability to promote Nrf2 degradation.
The KEAP1-Nrf2 pathway has attracted much attention not only for its role in oxidative stress, but also for its role in various diseases, including cancer, neurodegenerative diseases, and chronic diseases such as diabetes. Mutations or dysregulation of KEAP1 can lead to abnormal activation or inhibition of Nrf2, which can lead to pathogenesis. For example, in some cancers, KEAP1 mutations lead to constitutive activation of Nrf2, which enhances the detoxification capacity and resistance of cancer cells to chemotherapeutic drugs, thereby contributing to the survival and proliferation of cancer cells.
KEAP1 is a cytoplasmic protein that acts as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 results in the accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, researchers investigated whether Keap1 deficiency affects the endosomal-lysosomal pathway. Lysosomal-endosomal protein levels, lysosomal function, and autophagic activity were analyzed by using KEAP1 knockout cells (MEFs) and combining western blot analysis and fluorescence microscopy with fluorescence and pulse-chase assays. Loss of keap1 was found to downregulate protein levels and activity of proteinase D enzymes. In addition, KEAP1 deficiency also caused lysosomal alterations accompanied by accumulation of autophagosomes. These studies show that KEAP1 deficiency increases non-degradative lysosomes and identify a novel role for KEAP1 in lysosomal function, which may have therapeutic implications.
The results here show that despite the increase in the number of acidic vesicles, the lysosomal degradation capacity of Keap1 knockout cells was not significantly altered compared to control cells (Figure 1A). However, induction of autophagy during EBSS incubation did not increase the degradation rate of lysosomes in KEAP1-deficient cells. Electron microscopy analysis showed that Keap1 knockout cells had more vesicles in the cytoplasm than control cells. However, these compartments did not display electron-dense material (Figure 1B). Analysis of LC3 and p62 protein levels after RAPA and LLOMe treatment showed that both proteins accumulated in KEAP1-deficient cells (Figure 1C, D). This accumulation was more pronounced when lysosomes were damaged by LLOMe treatment.
Figure 1. KEAP1 knockout induces autophagic changes. (Uribe-Carretero, Elisabet, et al. 2022)
1. Cancer Research: The KEAP1-NRF2 pathway plays a critical role in the cellular response to oxidative stress and is frequently dysregulated in cancer. Using the KEAP1 knockout HeLa cell line can help researchers study the mechanisms by which loss of KEAP1 leads to cancer progression, angiogenesis, and resistance to chemotherapeutic drugs.
2. Drug Screening and Development: The KEAP1 knockout HeLa cell line provides a powerful model system for screening potential compounds that can modulate NRF2 activity, thereby aiding the identification of new therapeutic agents.
3. Oxidative Stress Research: Oxidative stress is associated with a variety of diseases, including neurodegenerative diseases, cardiovascular diseases, and metabolic syndrome. Researchers can use KEAP1 knockout HeLa cells to study how disruption of the KEAP1-NRF2 pathway affects cellular redox balance and oxidative stress responses.
4. Genetic and Epigenetic Regulation: The KEAP1 knockout HeLa cell line can be used to study the genetic and epigenetic mechanisms that regulate the KEAP1-NRF2 pathway. This includes understanding how gene expression and epigenetic modifications are altered in the absence of KEAP1 and how these changes affect cellular physiology and disease states.
5. Toxicology studies: KEAP1 knockout HeLa cells can be used to assess the effects of various toxins and environmental stressors on cells. By examining the differential activation of the NRF2 pathway in the absence of KEAP1, researchers can gain insight into cellular defense mechanisms against toxic stimuli and environmental pollutants.
6. Cellular senescence and aging: The KEAP1 knockout HeLa cell line can be used to study the role of the KEAP1-NRF2 pathway in cellular senescence and aging. Researchers can study how the loss of KEAP1 affects the aging process at the cellular level, including changes in cell morphology, function, and lifespan.
7. Inflammatory response: The NRF2 pathway, which is regulated by KEAP1, is involved in regulating inflammatory responses. By using KEAP1 knockout HeLa cells, researchers can explore how the KEAP1-NRF2 axis affects the expression of inflammatory cytokines and mediators, providing insight into inflammatory diseases.
For research use only. Not intended for any clinical use.
