Human SMAD4 Knockout Cell Line-HCT116
Cat.No. : CSC-RT0069
Host Cell: HCT116 Target Gene: SMAD4
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0069 |
| Cell Line Information | HCT116 -SMAD4 (-/-) is a cell line with a homozygous knockout of human SMAD4 |
| Target Gene | SMAD4 |
| Host Cell | HCT116 |
| Species | Human |
| 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. |
| 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
SMAD4, also known as Mothers Against Decapentaplegic Homolog 4, is a key protein in humans that plays a crucial role in the signaling pathways of the transforming growth factor-β (TGF-β) superfamily, which includes TGF-β, activins, and bone morphogenetic proteins (BMPs). SMAD4 plays important roles in a variety of cellular processes such as proliferation, differentiation, and apoptosis. Functionally, SMAD4 is a common mediator or "co-SMAD" in TGF-β signaling. Upon ligand binding, type I and type II TGF-β receptors form heterotetrameric complexes that phosphorylate receptor-regulated SMADs (R-SMADs), such as SMAD2 and SMAD3. Upon phosphorylation, these R-SMADs form a complex with SMAD4 and translocate to the nucleus, where they regulate the transcription of target genes by binding to specific DNA sequences or interacting with other transcription factors.
Notably, SMAD4 plays an important role in preventing tumor development. Its inactivation or mutation is often associated with various cancers, including pancreatic, colorectal, and gastric cancers. Germline mutations in SMAD4 are also associated with a genetic disorder called juvenile polyposis syndrome (JPS), which is characterized by the presence of numerous polyps in the gastrointestinal tract and an increased risk of malignant transformation.
SMAD4 loss-of-function mutations are frequently observed in colorectal cancer (CRC) and are considered drug targets for therapeutic development. Here, researchers performed a synthetic lethal drug screen using SMAD4-orthologous CRC cells and found that aurora kinase A (AURKA) inhibition was synthetically lethal together with SMAD4 loss. Inhibition of AURKA selectively suppressed the growth of SMAD4 knockout (SMAD4−/−) CRCs in vitro and in vivo. Mechanistically, SMAD4 negatively regulated AURKA levels, resulting in significant elevation of AURKA in SMAD4−/− CRC cells. Inhibition of AURKA resulted in G2/M cell cycle delay in SMAD4+/+ CRC cells but induced apoptosis in SMAD4−/− CRC cells. High levels of AURKA in SMAD4−/− CRC cells resulted in mitotic spindle abnormalities, leading to cellular aneuploidy. In addition, SMAD4−/− CRC cells expressed high levels of spindle assembly checkpoint (SAC) proteins, indicating that SAC is overactive. Silencing of key SAC proteins significantly rescued cell death caused by AURKA inhibition in SMAD4−/− cells, indicating that SMAD4−/− CRC cells are highly dependent on AURKA activity for mitotic exit and survival during SAC overactivity. This study demonstrates a unique synthetic lethal interaction between SMAD4 and AURKA and suggests that AURKA may be a potential drug target for SMAD4-deficient CRC.
Figure 1. Defective mitotic spindles and abnormal interphase nuclei formation in SMAD4-deficient CRC cells. a Immunofluorescence analysis of mitotic spindles and centrosomes stained with Hoechst33342 (HO33342, staining for DNA), α-tubulin antibody (staining for microtubule and spindles) and AURKA antibody (staining for centrosomes). Representative images of normal bipolar spindles and abnormal spindles in the mitotic phase of SMAD4 Knockout Cell Line-HCT116 (HCT116 SMAD4−/−) cells are shown. b HCT116 SMAD4+/+ and SMAD4−/− cells in the mitotic phase were analyzed for spindle morphology and spindle abnormality was quantified by counting cells with normal and abnormal spindles. c, d RT-qPCR data to analyze mRNA levels of MAD2L1, MPS1, BUB1, BUB1B and CDC20C in SMAD4-isogenic HCT116 (c) and DLD1 (d) cells are shown. e Western blot analysis of BUB1B in SMAD4- isogenic HCT116 and DLD1 cells is shown. f Immunofluorescence analysis of interphase nuclei. Representative images of normal nuclei and abnormal nuclei in HCT116 SMAD4−/− cells are shown. g Interphase nuclei in HCT116 SMAD4+/+ and SMAD4−/− cells were analyzed and the nuclear abnormality was quantified by counting cells with normal and abnormal nuclei. (Shi, Changxiang, et al. 2022)
The human SMAD4 knockout cell line - HCT116 is a powerful tool for studying a variety of biological and pathological processes. SMAD4 is a key component of the TGF-β signaling pathway and its disruption can lead to important insights into cancer biology, cell signaling, and therapeutic responses. Here are some of the key applications of this cell line:
Cancer Research: The SMAD4 knockout cell line - HCT116 is widely used in cancer research, especially in colorectal cancer research. By observing the behavior of these cells, researchers can gain insight into the role of SMAD4 in tumorigenesis, cell proliferation, and metastasis.
Signaling Pathway Studies: Since SMAD4 is a key mediator in the TGF-β pathway, knocking out this gene helps to elucidate its contribution to signal transduction and cellular responses.
Drug Development: HCT116 SMAD4 knockout cells play an important role in drug screening and development. By testing new compounds on these cells, scientists can evaluate the efficacy and potential side effects of candidate drugs targeting pathways and processes involving SMAD4.
Genetic Studies: Researchers can explore the downstream effects of SMAD4 loss and identify compensatory mechanisms that cells may adopt to cope with the loss of this gene.
Biomarker Discovery: This cell line can help identify new biomarkers for disease. By comparing analysis with wild-type cells, researchers can pinpoint specific proteins and genes that are differentially expressed due to SMAD4 loss, thereby finding potential new targets for diagnosis and treatment.
For research use only. Not intended for any clinical use.
