GRAMD1B Knockout SK-HEP-1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human liver adenocarcinoma cell line SK-HEP-1, engineered to disrupt the GRAMD1B gene locus. This pooled knockout model is designed for the study of non-vesicular cholesterol trafficking mechanisms at endoplasmic reticulum?Cplasma membrane (ER?CPM) contact sites, providing a heterogeneous population of edited cells suitable for bulk functional analyses without clonal selection. The product serves as a versatile tool for investigating lipid metabolic signaling in hepatocellular carcinoma (HCC) contexts, enabling researchers to interrogate GRAMD1B-dependent cholesterol sensing and its coupling to sterol regulatory element?Cbinding protein (SREBP) pathway activation.
SK-HEP-1 is an adherent liver adenocarcinoma cell line originally isolated from the ascitic fluid of a male patient, and it is widely employed as an HCC model due to its mixed epithelial and mesenchymal features. These cells co-express markers of both endothelial and hepatic origin, making them particularly useful for studies of tumor metastasis, angiogenic signaling, and acquired drug resistance. The line??s dual phenotype facilitates investigation into epithelial?Cmesenchymal transition dynamics and the contribution of metabolic reprogramming to malignant progression, providing a relevant background for dissecting the role of cholesterol homeostasis in liver cancer biology.
The GRAMD1B protein (also known as Aster-B) functions as a sterol transporter that shuttles cholesterol from the plasma membrane to the ER in a phosphatidylserine-dependent manner, acting as a sensor of membrane cholesterol levels. GRAMD1B interacts with ER-resident VAP proteins (VAPA and VAPB) as well as other lipid transfer proteins such as OSBP and CERT, and it is functionally linked to NPC1-dependent cholesterol export from lysosomes. In response to plasma membrane cholesterol enrichment, GRAMD1B transports excess cholesterol to the ER, where it suppresses the SREBP pathway by promoting Insig-mediated retention of SREBP2. Knockout of GRAMD1B disrupts this feedback, leading to ER cholesterol depletion and consequent activation of SREBP2 and SREBP1, which transcriptionally upregulate genes such as LDLR, HMGCR, and ABCA1. Additionally, GRAMD1B loss alters downstream inflammatory and ER stress responses, including modulation of IL-6, TNF-??, CHOP, and BiP expression.
In the SK-HEP-1 HCC model, ablation of GRAMD1B is predicted to perturb intracellular cholesterol distribution, thereby influencing cell proliferation, migration, and sensitivity to chemotherapeutic agents through deregulation of SREBP and associated Akt/mTOR signaling nodes. This knockout model is particularly relevant for investigating the intersection of hepatic cholesterol metabolism and oncogenesis, as dysregulated lipid handling is a hallmark of non-alcoholic fatty liver disease, metabolic syndrome, and atherosclerosis. By engineering GRAMD1B disruption in cells that already exhibit altered cholesterol flux, researchers can explore how ER?CPM contact site dysfunction contributes to lipotoxicity, inflammatory cytokine secretion, and therapeutic evasion in liver cancer.
Core research applications include quantitative analysis of SREBP processing by western blotting, visualization of cellular cholesterol distribution using filipin staining, and transcriptional profiling of cholesterol-related genes via RT-qPCR. The model supports functional cholesterol efflux assays, co-immunoprecipitation studies with VAPA or VAPB to probe contact site integrity, and immunofluorescence-based assessment of ER?CPM apposition. Migration chamber assays and cell proliferation measurements can further delineate phenotypic consequences of GRAMD1B loss. For additional details or technical inquiries regarding this knockout cell product, please contact Ascent Research.
