ATP9A Knockout HCT 116 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HCT 116 colorectal carcinoma cell line, engineered to disrupt the ATP9A gene. This product provides a pooled population of gene-edited cells for investigating ATP9A loss-of-function effects in endolysosomal trafficking, lipid asymmetry, and associated signaling pathways. The polyclonal format captures a range of editing events, enabling robust population-level analyses without clonal selection.
The HCT 116 cell line is a widely used epithelial model of colorectal adenocarcinoma, characterized by KRAS G13D and PIK3CA H1047R oncogenic mutations, microsatellite instability (MSI-H) due to MLH1 promoter hypermethylation, and elevated ??-catenin signaling. These genetic features render HCT 116 particularly valuable for studying oncogenic signaling, DNA mismatch repair defects, and drug response mechanisms, including chemoresistance and targeted therapy sensitivity.
ATP9A encodes a P4-ATPase phospholipid flippase that specifically translocates phosphatidylserine from the exoplasmic to the cytoplasmic leaflet of endolysosomal membranes. Its activity is regulated by CDC50A (TMEM30A) and CDC50B (TMEM30B) accessory subunits, intracellular Ca2?, and membrane lipid composition. ATP9A is transcriptionally controlled by TFEB and functions within endocytic trafficking and autophagy pathways, interacting with Rab5, Rab7, and ESCRT machinery. Downstream, ATP9A-mediated phospholipid redistribution is essential for endosomal cargo sorting (e.g., EGFR), proper lysosomal acidification, cathepsin maturation, and mTORC1 signaling activation. Loss of ATP9A disrupts membrane lipid asymmetry, leading to impaired lysosomal degradation, defective autophagic flux, and altered mTORC1 activity.
In the HCT 116 background, ATP9A knockout offers a unique investigative tool for dissecting how endolysosomal dysfunction intersects with colorectal cancer biology. The cell line’s MSI-H status and RAS/PI3K pathway activation provide a platform to examine interactions between lipid flippase activity and oncogenic signaling, particularly mTORC1-driven growth and autophagy-mediated survival under stress. ATP9A loss may also modulate responses to lysosomotropic agents or autophagy-modulating drugs, as well as influence antigen presentation via exosome secretion, relevant to immunotherapy research. Moreover, the model enables exploration of neurodevelopmental disorder-associated mechanisms in a cancer-relevant context, bridging research areas such as lysosomal storage-like dysfunctions and tumorigenesis.
These polyclonal knockout cells are well-suited for a range of assays including Western blotting, immunofluorescence, and RT-qPCR for expression profiling; flow cytometry with Annexin V to assess phosphatidylserine externalization; lysosomal pH measurements via LysoTracker staining; autophagic flux monitoring by LC3 turnover; mTOR phospho-signaling analysis; and RNA-seq for transcriptomic profiling. They facilitate functional genomics screens to identify flippase modulators and validate drug targets in endolysosomal disorders. The pooled population enables high-content screening for compounds that rescue ATP9A-deficient phenotypes. For further information regarding this product, customization options, or technical support, please contact Ascent Research.
