ACOT9 Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-mediated gene-disrupted polyclonal cell population generated from the HT29 human colorectal adenocarcinoma line. This loss-of-function model eliminates ACOT9 enzymatic activity, enabling study of acyl-CoA thioesterase 9 in its native context. The polyclonal format provides a heterogeneous knockout population suitable for metabolic and signaling analyses, without implying clonal selection or homozygous disruption. Researchers can use these cells to dissect ACOT9’s role in fatty acid handling and downstream transcriptional programs.
HT29 cells were originally established from a primary colon adenocarcinoma of a 44-year-old female. These adherent, epithelial-like cells are widely used as an intestinal epithelial and colorectal cancer model. Their well-characterized growth, tumorigenic potential, and responsiveness to metabolic perturbations make them an ideal background for interrogating lipid-modifying enzymes. The epithelial origin retains relevant polarity, adhesion, and membrane trafficking machinery essential for lipid signaling studies.
ACOT9 hydrolyzes mitochondrial long-chain acyl-CoAs to free fatty acids and CoA, modulating intracellular metabolite pools. This reaction is a key node connecting fatty acid oxidation and nuclear receptor signaling. ACOT9 expression is regulated by PPAR?? and PPAR??, with coactivator PGC-1?? enhancing induction upon fatty acid stimulation. Released fatty acids serve as ligands for PPAR transcription factors, which heterodimerize with RXR?? to drive target genes including UCP3. ACOT9 thus controls acyl-CoA flux into ??-oxidation, where CPT1, ACADs, ECHS1, and HADHA process substrates, while FABP1 facilitates fatty acid trafficking. This network positions ACOT9 as a critical regulator of mitochondrial energy metabolism and lipid-mediated transcription.
In colorectal cancer, ACOT9 knockout disrupts the balance between fatty acid oxidation and anabolic lipid utilization. HT29 cells undergo metabolic reprogramming to support proliferation; loss of ACOT9 may elevate long-chain acyl-CoA levels, impairing ??-oxidation and altering PPAR-dependent gene programs governing survival, proliferation, and inflammation. This model offers a platform to study how lipid thioesterase activity influences oncogenic metabolism and reveals metabolic vulnerabilities in obesity- and metabolic syndrome-associated cancers.
These polyclonal knockout cells are suitable for fatty acid oxidation assays and Seahorse mitochondrial stress tests to quantify oxidative metabolic changes. Western blotting and RT-qPCR validate ACOT9 disruption and assess downstream metabolic gene expression; lipidomics profiles shifts in acyl-CoA and free fatty acid pools. PPAR luciferase reporter assays measure transcriptional activity, and proliferation assays under lipid deprivation reveal metabolic dependencies. This versatile tool supports drug screening for metabolic targets and mechanistic studies of lipid signaling in cancer. For further details, please contact Ascent Research.
