The ATG14 Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal population derived from the human colorectal adenocarcinoma HT-29 cell line, engineered to disrupt the ATG14 gene. This product provides a versatile loss-of-function model in which ATG14-mediated signaling is abrogated across a heterogeneous cell pool, enabling functional studies of autophagy in an epithelial cancer context. Through CRISPR/Cas9-mediated gene disruption, the cells serve as a critical tool for investigating the roles of ATG14 in autophagosome biogenesis, nutrient sensing, and colorectal cancer progression without the constraints of clonal selection.
HT-29 is a widely utilized human epithelial colorectal adenocarcinoma cell line derived from a primary tumor, exhibiting adherent epithelial morphology and retaining key features of intestinal epithelial cells. These cells are extensively employed in cancer biology for studying colorectal adenocarcinoma pathophysiology, drug development, and intestinal epithelial barrier function. The HT-29 background provides a relevant and physiologically meaningful environment for dissecting autophagy-related mechanisms given its expression of core autophagy machinery and its responsiveness to metabolic and pharmacological modulators commonly used in oncology research.
ATG14 functions as a pivotal scaffold protein within the autophagy-specific class III phosphatidylinositol 3-kinase (PI3K) complex I, also known as the VPS34 complex. It physically interacts with BECN1, PIK3C3 (VPS34), and PIK3R4 (p150) to form the core complex, and its membrane-targeting BATS domain directs the holoenzyme to the phagophore. ATG14 is activated downstream of the ULK1 kinase complex and is regulated by nutrient-sensing pathways, being positively modulated by AMPK and negatively controlled by MTORC1 through phosphorylation events. Once localized, ATG14 stimulates production of phosphatidylinositol 3-phosphate (PI3P), which recruits downstream effectors such as WIPI2 and facilitates the lipidation and membrane conjugation of MAP1LC3B and GABARAP family proteins, thereby driving autophagosome nucleation and expansion. Additional interacting partners include ATG13 and RB1CC1, placing ATG14 at the nexus of autophagic initiation.
In the context of HT-29 colorectal adenocarcinoma cells, ATG14 knockout disrupts a fundamental node of the autophagy pathway, which is often dysregulated in colorectal cancer and associated with tumor progression, chemotherapy resistance, and inflammatory bowel diseases like Crohn’s disease. This model enables direct assessment of how loss of ATG14-dependent autophagy influences tumor cell survival under nutrient deprivation, hypoxic stress, or chemotherapeutic challenge, making it particularly valuable for dissecting resistance mechanisms to agents such as 5-fluorouracil or oxaliplatin. Furthermore, the epithelial nature of HT-29 cells allows investigation of autophagy’s role in maintaining intestinal barrier integrity and its contribution to inflammation-driven carcinogenesis.
Research applications for these polyclonal knockout cells are diverse and include detailed autophagy flux analysis using chloroquine to monitor MAP1LC3B turnover, immunofluorescence microscopy to quantify LC3 puncta formation, and Western blotting to detect lipidated LC3B-II as a hallmark of autophagic activity. Complementary assays such as cell viability measurements, colony formation assays, RT-qPCR for ATG14 expression validation, and flow cytometry-based autophagy detection expand the utility to phenotypic screening of autophagy modulators, metabolism studies, and stress response pathways. This product is suited for laboratories investigating autophagy??s role in colorectal cancer or screening for novel therapeutic targets. For additional product specifications and support, please contact Ascent Research.
