The GRAMD1B Knockout A-549 Polyclonal Cells are CRISPR/Cas9-edited polyclonal knockout populations, generated from the A-549 cell line to disrupt GRAMD1B function. This heterogeneous cell pool enables loss-of-function analysis of the GRAMD1B gene, which encodes a cholesterol transporter at ER-PM contact sites. It provides a flexible model for studying sterol sensing and non-vesicular cholesterol trafficking in a lung adenocarcinoma background.
A-549 cells are human lung adenocarcinoma epithelial cells harboring an activating KRAS mutation, widely used in cancer biology for metastasis, drug screening, and oncogenic signaling research. Their epithelial nature and well-documented lipid metabolism make them ideal for investigating cholesterol handling in KRAS-driven tumors. The knockout of GRAMD1B in this line allows dissection of how cancer cells regulate lipid distribution through membrane contact site-dependent mechanisms.
GRAMD1B acts as a sterol sensor at the plasma membrane, binding accessible cholesterol and PI(4,5)P2, and interacting with VAPA/VAPB to transport cholesterol to the endoplasmic reticulum. This transport modulates SREBP cleavage, suppressing cholesterogenic gene transcription (LDLR, HMGCR) when cholesterol is abundant. GRAMD1B is regulated by LXR, oxysterols, and cellular cholesterol levels, and influences downstream processes such as cholesterol esterification by ACAT, ER stress responses, and lipid droplet formation. It functions alongside Aster family members but exhibits unique regulatory properties.
In A-549 cells, KRAS oncogenic signaling drives increased cholesterol and lipid demand; disruption of GRAMD1B is expected to impair ER cholesterol sensing, leading to constitutive SREBP activation and altered lipid homeostasis. This model is valuable for studying metabolic adaptation in cancer, drug resistance linked to lipid reprogramming, and potential synthetic lethal interactions with cholesterol pathway inhibitors. The polyclonal nature allows assessment of GRAMD1B function without clonal artifacts.
Applications include filipin staining to map cholesterol distribution, cholesterol efflux/uptake assays, western blotting for SREBP processing, RT-qPCR of cholesterogenic genes, co-immunoprecipitation with VAP proteins, and viability assays under lipid deprivation. These assays support investigations into membrane contact site biology, KRAS-lipid crosstalk, and the role of sterol transport in metastasis. For further details or custom applications, please contact Ascent Research.
