The ATP6V0A1 Knockout HeLa Polyclonal Cells are a mixed population of HeLa cells modified by CRISPR/Cas9 to disrupt the ATP6V0A1 gene, encoding the V-ATPase V0 domain a1 subunit. This polyclonal format provides a heterogeneous knockout model without clonal selection, suited for investigating V-ATPase-dependent acidification and associated cellular functions.
The HeLa host line is a cervical adenocarcinoma epithelial cell model, immortalized by HPV18, with p53 and Rb inactivation, aneuploidy, and high proliferative capacity. Widely employed in cancer and cell biology research, HeLa cells offer robust transfection efficiency and well-defined signaling pathways, making them ideal for genetic perturbation studies.
ATP6V0A1 encodes the a1 subunit of V-ATPase, essential for proton pumping and acidification of endosomes, lysosomes, and other organelles. This proton gradient enables mTORC1 activation on the lysosomal surface via the Ragulator-Rag GTPase-LAMTOR1 complex, linking nutrient sensing to cell growth. ATP6V0A1 interacts with additional V-ATPase subunits (ATP6V0C, ATP6V1A), accessory proteins (ATP6AP1/2), and the small GTPases Rab7 and Arf6, mediating endosomal trafficking and acidification. Upstream, its activity is regulated by mTORC1, AMPK, and growth factors (EGF, insulin) in response to amino acid and glucose levels. Downstream, ATP6V0A1 function impacts mTORC1-mediated phosphorylation of p70 S6 kinase, TFEB nuclear translocation governing lysosomal biogenesis and autophagy gene expression, and activation of lysosomal cathepsins. Consequently, knockout impairs autophagic flux and endosomal sorting.
In HeLa cells, ATP6V0A1-dependent acidification is central to the aggressive tumor cell phenotype, influencing growth factor signaling, metabolic adaptation, and resistance. The polyclonal knockout population allows direct analysis of how disrupted V-ATPase function alters intracellular pH, endocytic trafficking, and autophagy in a heterogeneous cancer cell background. This model avoids potential biases from single-cell clones and better recapitulates the diversity of responses within a tumor cell population, offering a robust platform for studying organellar acidification in cancer.
These cells are suitable for Western blotting and RT-qPCR to confirm knockout, immunofluorescence with LysoTracker for lysosomal pH assessment, and functional assays such as phospho-p70 S6K immunoblotting for mTORC1 activity, LC3B-II turnover for autophagic flux, and cathepsin activity measurements. Applications span investigations of endocytosis, autophagy, mTOR signaling, cancer metabolism, drug resistance, and pH-dependent metastasis. For inquiries, please contact Ascent Research.
