The HECTD3 Knockout SK-HEP-1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal cell population derived from the human SK-HEP-1 hepatocellular carcinoma line, featuring targeted disruption of the HECTD3 gene. This knockout pool provides a heterogeneous mixture of HECTD3 loss-of-function alleles, circumventing clonal bias and enabling robust functional studies. The cells are designed to ablate HECTD3 E3 ubiquitin ligase activity, facilitating direct investigation of its roles in ubiquitin-dependent signaling, apoptosis, and intracellular trafficking.
The parental SK-HEP-1 cell line is an immortalized epithelial line originally isolated from the ascites of a patient with liver adenocarcinoma. Widely employed as a model for hepatocellular carcinoma (HCC), SK-HEP-1 cells retain key oncogenic properties and are extensively used to study HCC cell proliferation, migration, apoptosis, and drug responses. Their well-characterized signaling networks and genetic tractability make them a suitable platform for dissecting gene function in liver cancer pathogenesis.
HECTD3 encodes an E3 ubiquitin ligase that cooperates with E2 conjugating enzymes of the UBE2D family to ubiquitinate substrate proteins, most notably caspase-8 (CASP8) and MALT1. Modification of CASP8 by HECTD3 influences activation of the caspase cascade involving CASP3 and BID, thereby regulating extrinsic apoptosis. Ubiquitination of MALT1 promotes NF-??B signaling through the IKBKG/NF-??B axis, controlling expression of pro-survival genes. Additionally, HECTD3 participates in DNA damage responses downstream of ATM/ATR and p53, and contributes to endosomal trafficking via its interaction with STX8 and the SNARE complex. Upstream, HECTD3 expression is induced by p53, TNF??, DNA damage, and ER stress, positioning it at the nexus of multiple stress-responsive pathways.
In SK-HEP-1 cells, knockout of HECTD3 disrupts ubiquitination of caspase-8 and MALT1, which may sensitize cells to apoptosis, alter NF-??B-dependent transcription, and modify responses to genotoxic stress. This polyclonal knockout model is expected to exhibit defective endosomal trafficking and altered cell migration, providing a powerful tool to dissect HECTD3-dependent mechanisms that sustain HCC viability, drug resistance, and metastatic dissemination.
Researchers can utilize these cells for diverse assays including co-immunoprecipitation and Western blotting to assess ubiquitination of endogenous substrates (CASP8, STX8, MALT1), RT-qPCR to quantify downstream gene changes, and immunofluorescence to visualize ??H2AX foci or STX8 localization. Functional studies may involve apoptosis measurement via Annexin V/PI staining, proliferation analysis by CCK-8 or clonogenic assay, and Transwell migration/invasion tests. Transcriptomic profiling by RNA-seq and drug sensitivity experiments with sorafenib or cisplatin further expand the utility of this knockout population. For additional information or technical support regarding this product, please contact Ascent Research.
