The DMXL1 Knockout SK-HEP-1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population targeting the DMXL1 gene in the SK-HEP-1 human hepatic adenocarcinoma cell line. This loss-of-function model provides a genetically disrupted DMXL1 background without clonal selection, enabling the study of heterogeneous cellular responses to DMXL1 ablation. The polyclonal format preserves the complexity of the CRISPR-edited pool, suitable for pooled screening and population-level assays.
SK-HEP-1 is an epithelial cell line derived from a human hepatic adenocarcinoma, commonly used as a model for liver cancer metastasis and angiogenesis. These cells exhibit robust growth and are amenable to genetic manipulation, making them a valuable platform for investigating signaling pathways implicated in hepatocellular carcinoma progression.
DMXL1, also known as rabconnectin-3 alpha, functions as a scaffold protein that bridges Rab3 GTPase-activating protein (Rab3 GAP) and the V-ATPase proton pump. This molecular scaffolding coordinates vesicular acidification and exocytosis by regulating Rab3 GTPase activity and V-ATPase assembly. Critically, DMXL1 is essential for amino acid-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) through the Ragulator-Rag GTPase axis. Upstream signals such as amino acid availability and growth factor receptors converge on DMXL1-containing complexes at the lysosomal surface, where DMXL1 facilitates the interaction between V-ATPase, Ragulator (LAMTOR1-5), and Rag GTPases. Downstream, DMXL1-dependent mTORC1 activation leads to phosphorylation of S6K1 and 4E-BP1, promoting protein synthesis and cell growth. Disruption of DMXL1 uncouples lysosomal acidification from mTORC1 signaling, impairing downstream effectors and potentially altering autophagy, as monitored by LC3B puncta formation.
In the SK-HEP-1 hepatocellular carcinoma context, DMXL1 knockout provides a direct tool to dissect its role in liver cancer cell proliferation, lysosomal function, and mTORC1-driven metabolism. The DMXL1-null SK-HEP-1 polyclonal cells enable examination of how loss of this scaffold impacts V-ATPase-dependent acidification and mTORC1 localization to lysosomes, processes frequently dysregulated in cancer. This model is particularly relevant for studying mechanisms of resistance or sensitivity to mTOR pathway inhibitors such as rapamycin, as DMXL1 may modulate the cellular response to such agents.
Researchers can employ these DMXL1 knockout polyclonal cells in a range of functional assays. Western blotting for phosphorylated S6K1 and 4E-BP1 reveals mTORC1 activity status, while immunofluorescence for LC3B allows assessment of autophagic flux. Lysosomal pH measurements and mTORC1 lysosomal translocation assays provide insights into V-ATPase function and Ragulator-dependent signaling. Proliferation and colony formation assays, coupled with co-immunoprecipitation of DMXL1-V-ATPase complexes, can elucidate the role of DMXL1 in cell growth and interactome dynamics. Additionally, these cells are suitable for drug sensitivity testing with rapamycin and other mTOR inhibitors. For further information or technical support, please contact Ascent Research.
