The ATG4B Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell pool in which the autophagy-related cysteine protease ATG4B has been disrupted. Derived from the HT29 colorectal adenocarcinoma cell line, this heterogeneous population provides a stable loss-of-function model for probing autophagy mechanisms. The polyclonal format overcomes clonal selection biases, offering a robust system for functional genomics and drug discovery applications focused on ATG4B-dependent pathways.
HT29 is a widely employed human colorectal adenocarcinoma epithelial line, originally isolated from a 44-year-old female patient. These adherent cells retain key oncogenic signaling features, including active Wnt/??-catenin and MAPK pathways, and serve as a standard model for intestinal epithelial biology and colorectal cancer. Their use facilitates the study of tumor cell-autonomous processes such as proliferation, drug sensitivity, and metabolic adaptation under stress.
ATG4B functions as a dual-activity cysteine protease in autophagy. It primes pro-LC3 (MAP1LC3B) by C-terminal cleavage to generate LC3-I, and later deconjugates LC3-II from autophagosomal membranes to recycle LC3. ATG4B is regulated by upstream mTOR, ROS, FOXO3, and TFEB signals. It targets MAP1LC3B and GABARAP family proteins, interacting with ATG7 and ATG3 in the conjugation cascade. Disruption of ATG4B impairs LC3-I to LC3-II conversion and blocks autophagosome maturation, attenuating autophagic flux.
In HT29 colorectal cancer cells, ATG4B knockout provides a critical tool to dissect autophagy??s contribution to tumor survival, chemoresistance, and proliferation. Colorectal tumors exploit autophagy for metabolic fitness and apoptosis evasion, and ATG4B is recognized as a potential therapeutic target. The polyclonal knockout population enables assessment of ATG4B-dependent functions without clonal artifacts, supporting robust analyses of autophagy-mediated responses to nutrient deprivation, hypoxia, and drug treatment. This model is ideal for studying the crosstalk between autophagy and oncogenic pathways, such as mTOR signaling, and for evaluating ATG4B inhibitors.
Key applications include monitoring autophagy flux via Western blotting for LC3-I/II conversion, immunofluorescence detection of LC3 puncta after Bafilomycin A1 treatment, and cell viability assays under stress. The cells are suited for colony formation, drug sensitivity profiling, and RT-qPCR analysis of autophagy gene expression. Researchers can use this model to investigate ATG4B-dependent autophagosome formation, explore compensatory mechanisms in the ATG8 conjugation system, and study autophagy-related cell death in colorectal cancer. For further details, contact Ascent Research.
