The ATG4A Knockout SK-HEP-1 Polyclonal Cells product supplies a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human SK-HEP-1 liver sinusoidal endothelial-like cell line. This pool harbors a diverse array of ATG4A gene disruptions, generating a loss-of-function model that eliminates the biases associated with monoclonal expansion. It is optimized for investigations into autophagy regulation and its intersection with endothelial biology and liver cancer pathogenesis.
The parental SK-HEP-1 cell line was originally isolated from the ascites fluid of a 52-year-old male patient with liver adenocarcinoma and has been characterized to exhibit endothelial features, including the expression of von Willebrand factor, uptake of acetylated low-density lipoprotein, and formation of capillary-like tubes in vitro. This line recapitulates key functional characteristics of liver sinusoidal endothelial cells, such as filtration, endocytosis, and participation in hepatic immune regulation. Its unique adenocarcinoma-derived, endothelial-like phenotype provides a robust platform for studying liver cancer cell biology, tumor microenvironment interactions, and sinusoidal endothelial cell physiology.
The ATG4A gene encodes a cysteine protease that primes pro-LC3 and pro-GABARAP proteins by exposing a C-terminal glycine for phosphatidylethanolamine conjugation, and also delipidates LC3-PE to recycle autophagy machinery during autophagosome maturation. ATG4A activity is controlled by the MTOR-AMPK-TFEB-FOXO3 regulatory network, and it functions in concert with ATG7, ATG3, and the ATG5-ATG12-ATG16L1 complex during LC3 lipidation. Its substrates LC3A, LC3B, GABARAP, and GABARAPL1 are incorporated into nascent autophagosomes, where the PI3K-III complex (Beclin1-VPS34) generates PI3P to facilitate membrane association. In addition, p62/SQSTM1 links polyubiquitinated cargo to LC3 for selective degradation. Consequently, ATG4A knockout ablates autophagosome formation and flux.
In the SK-HEP-1 background, ATG4A deficiency disrupts autophagy-dependent quality control, rendering cells more sensitive to proteotoxic, metabolic, and chemotherapeutic stress. This model is particularly suited to elucidating the dual roles of autophagy in hepatocellular carcinoma, where it can suppress tumor initiation by eliminating damaged organelles or facilitate tumor survival under therapeutic pressure. The endothelial-like nature of SK-HEP-1 cells also permits dissection of autophagy??s contribution to sinusoidal endothelial homeostasis, including fenestration stability and lipid droplet handling.
Typical applications include autophagy flux assays using Western blotting for LC3-II accumulation in the presence of bafilomycin A1, immunofluorescence for LC3 puncta quantification, and RT-qPCR profiling of autophagy-related gene expression. Co-immunoprecipitation can validate ATG4A??s interactions with ATG7, ATG3, LC3B, or GABARAP. The cells are also suitable for migration and apoptosis flow cytometry assays under autophagy-modifying conditions, as well as for drug screening campaigns targeting autophagy modulators. For additional technical details or a quotation, please contact Ascent Research.
