Human BAK1 Knockout Cell Line-HeLa
Cat.No. : CSC-RT1709
Host Cell: HeLa Target Gene: BAK1
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1709 |
| Cell Line Information | A stable cell line with a homozygous knockout of human BAK1 using CRISPR/Cas9. |
| Target Gene | BAK1 |
| Host Cell | HeLa |
| Shipping | 10^6 cells/tube |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene ID | 578 |
| 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
The BAK1 gene, formally known as BCL2 antagonist/killer 1, is a member of the BCL-2 gene family and plays a key role in regulating apoptosis, a programmed cell death necessary for maintaining cellular homeostasis. BAK1 is located on chromosome 6p21.2 and produces a protein that localizes to the mitochondrial membrane. Upon activation, BAK1 undergoes a conformational change that causes it to oligomerize and form pores on the outer mitochondrial membrane. This process leads to the release of cytochrome c and other pro-apoptotic factors from the mitochondria into the cytosol, thereby activating caspases, the executioners of apoptosis. This pore-forming activity is a key step in the intrinsic pathway of apoptosis, making BAK1 a central mediator of cell death.
The regulation of BAK1 is complex and occurs at multiple levels, including transcriptional, post-transcriptional, and post-translational modifications. Various proteins and signaling pathways can influence BAK1 activity. For example, anti-apoptotic proteins such as BCL-2 and BCL-XL can bind to BAK1 and inhibit its pro-apoptotic function, thereby preventing apoptosis induction under specific conditions. Studies have shown that BAK1 dysregulation is associated with many diseases, especially cancer. Overexpression or underregulation of BAK1 leads to excessive cell death, resulting in degenerative diseases, while its downregulation or inhibition leads to uncontrolled cell proliferation and resistance to apoptosis, which are hallmarks of cancer. Therefore, understanding the precise mechanisms that control BAK1 activity is important for developing therapeutic strategies.
BH3 analogs are a promising tool in cancer therapy. Recently, drugs targeting Mcl-1 proteins entered clinical trials, and many studies have focused on investigating their activity in various preclinical models. Here, researchers studied two BH3 analogs targeting Mcl-1, A1210477 and S63845, and found that although both drugs interacted with the target, their efficacy at targeted doses was different. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. In cells that acquired resistance to Mcl-1 inhibition, Bcl-xL protein levels increased. Cell lines sensitive to S63845 exhibited low Bcl-xL expression. Tumor tissue from patients with lung adenocarcinoma was characterized by decreased Bcl-xL and increased Bak mRNA and protein levels. Surprisingly, in Bak knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in significant cell death, arguing against a sole role for Bak in the studied phenomenon. These studies suggest that Bak and Bcl-xL are cofactors for sensitivity and resistance to Mcl-1 inhibition, respectively.
The analysis showed that BAK1 knockout did not rescue HeLa and H23 cells from the cytotoxic effects of the combination of A1331852 and S63845. In fact, caspase-3 processing and PARP cleavage were already intense in both control and BAK1 knockout cells after 4 h of incubation (Figure 1A,C). In agreement with the Western blot results, flow cytometry showed a high rate of cell death in HeLa cells (Figure 1B). Thus, even in the absence of Bak, simultaneous targeting of Bcl-xL and Mcl-1 exhibited high cytotoxicity.
Figure 1. Analysis of the efficiency of concomitant inhibition of Mcl-1 and Bcl-xL in Bak1 knockout cells. (A,C) WB analysis of HeLa (A) or H23 (C) cells with or without knockout of BAK1. (B) FACS analysis of HeLa cells with or without knockout of BAK1. (Senichkin V V, et al. 2021)
Here are some potential applications of human BAK1 knockout cell lines:
Apoptosis Research: BAK1 (BCL2 antagonist/killer 1) is an important pro-apoptotic member of the BCL-2 family of proteins. Knockout HeLa cells that lack BAK1 can be used to study pathways and mechanisms of apoptosis.
Cancer Research: Since HeLa cells are derived from cervical cancer, BAK1 knockout in these cells helps understand the role of BAK1 in cancer cell biology. Researchers can study how loss of BAK1 affects tumor growth, cancer cell survival, and resistance to chemotherapeutic drugs.
Drug Screening: BAK1 knockout HeLa cells are valuable in high-throughput drug screening assays. These cells can be used to identify compounds that selectively induce apoptosis in BAK1-deficient cells. This can help develop drugs that are effective against cancer cells with similar genetic characteristics.
Mitochondrial Dysfunction Research: BAK1 is involved in mitochondrial-mediated apoptosis. BAK1 knockout HeLa cells can be used to study how loss of BAK1 affects mitochondrial function, dynamics, and interactions with other proteins.
Gene Regulation and Signaling: Studying BAK1 knockout cells can provide valuable information about how this gene affects various signaling pathways and gene expression patterns. It helps identify downstream targets and partners of BAK1, thus elucidating its broader role in cell biology.
For research use only. Not intended for any clinical use.
