Human ATF4 Knockout Cell Line-Hela
Cat.No. : CSC-RT2793
Host Cell: Hela Target Gene: ATF4
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2793 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human ATF4 using CRISPR/Cas9. |
| Target Gene | ATF4 |
| Host Cell | Hela |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid Nitrogen |
| 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
ATF4, or activating transcription factor 4, is a key protein involved in the cellular stress response. ATF4 is synthesized in response to stress signals through a mechanism involving the integrated stress response (ISR) pathway. Under normal conditions, ATF4 mRNA transcription is repressed by the upstream open reading frame (uORF), which inhibits its translation. However, during stress, phosphorylation by eukaryotic initiation factor 2 alpha (eIF2α) shifts the translation start site, allowing the production of ATF4 protein. Once produced, ATF4 translocates to the nucleus, where it binds to specific DNA sequences and activates transcription of target genes. This regulation is essential for cellular adaptation during environmental and physiological stresses, such as nutrient deprivation, hypoxia, and endoplasmic reticulum (ER) stress.
In the context of metabolic diseases, ATF4 helps manage the cellular response to amino acid limitation by activating genes responsible for amino acid transport and synthesis. Deficiency or dysregulation of ATF4 can lead to metabolic imbalances and contribute to diseases such as diabetes. In addition, ATF4 is involved in bone biology, affecting the differentiation of osteoblasts (cells responsible for bone formation). Therefore, mutations or abnormal expression of ATF4 can affect bone density and lead to diseases such as osteoporosis. ATF4 is also important in neurodegenerative diseases. Sustained activation of ATF4 can lead to apoptosis of nerve cells and aggravate neurodegeneration. In cancer, ATF4 has a dual role. On the one hand, it can induce apoptosis of cancer cells under severe stress and act as a tumor suppressor. On the other hand, ATF4 can also support tumor survival by upregulating genes involved in antioxidant responses and autophagy, helping cancer cells tolerate the harsh tumor microenvironment.
Mitochondrial respiration is essential for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors such as aspartate, an essential substrate for nucleotide synthesis. Here, researchers show that, in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. In the context of ETC inhibition, exogenous asparagine restores proliferation, ATF4 and mTORC1 activity, and mTORC1-dependent nucleotide synthesis, suggesting that asparagine relays active respiration to ATF4 and mTORC1. Finally, researchers show that the combination of the ETC inhibitor metformin (to limit tumor asparagine synthesis) with either asparaginase or dietary asparagine restriction (to limit tumor asparagine consumption) effectively inhibits tumor growth in multiple mouse cancer models.
The extent to which exogenous asparagine limited ATF4 activation by ETC inhibition correlated with the ability of asparagine to restore mTORC1 activity: no rescue of mTORC1 by asparagine was observed with antimycin A, where asparagine resulted in only partial rescue of ATF4, whereas in the presence of complex I and complex V inhibition, asparagine fully restored mTORC1 activity, whereas ATF4 activation was completely blocked (Figure 1A, B). Thus, the researchers proposed that ATF4 activation may contribute to the reduced mTORC1 activity resulting from respiratory impairment. To test this possibility, they engineered ATF4 knockout HeLa cells to constitutively express ASNS, thereby uncoupling ASNS activity and asparagine synthesis from ATF4 expression. These data indicate that in the absence of ATF4, mTORC1 activity is insensitive to ETC inhibition (constitutively active) (Figure 1E), raising the possibility that asparagine signals respiratory activity to mTORC1 at least in part through ATF4.
Figure 1. Asparagine relays mitochondrial respiration to ATF4 and mTORC1. (Krall A S, et al., 2021)
Cancer Research: ATF4 (activating transcription factor 4) plays a key role in cell proliferation and survival under stress conditions. By using ATF4 knockout Hela cells, researchers can study the pathways and mechanisms by which ATF4 affects cancer cell growth and survival.
Stress Response Research: ATF4 is a master regulator of the integrated stress response (ISR). Knockout cell lines allow for detailed studies of cellular responses to different types of stress, including endoplasmic reticulum (ER) stress and oxidative stress. This helps understand stress-related diseases such as neurodegenerative diseases and diabetes.
Gene Regulation and Expression: Scientists can use ATF4 knockout Hela cells to understand the role of ATF4 in regulating other genes. By comparing gene expression profiles between knockout and wild-type cells, researchers can identify downstream targets of ATF4 and elucidate its regulatory networks.
Drug Testing and Development: These knockout cell lines can be used for high-throughput screening of potential drugs. By observing the effects of various compounds on ATF4-deficient cells, researchers can identify drugs that may be particularly effective or discover unexpected effects on cellular stress pathways.
Metabolic Research: ATF4 is involved in metabolic pathways including amino acid metabolism and autophagy. Utilizing ATF4 knockout Hela cells facilitates the study of these metabolic processes, which is critical for understanding metabolic disorders and developing appropriate treatments.
For research use only. Not intended for any clinical use.
