TMEM97 Knockout U266 Cell Line

The TMEM97 Knockout U266 Cell Line is a CRISPR/Cas9-edited knockout cell line that disrupts TMEM97 expression in the human U266 B lymphocyte line. This loss-of-function model enables precise study of sigma-2 receptor biology in multiple myeloma, avoiding off-target effects of pharmacological ligands. It provides a stable system for investigating cholesterol homeostasis, autophagy, and apoptosis in a disease-relevant context.

U266 is an EBV-negative multiple myeloma cell line derived from a 53-year-old male patient. These IgE-secreting plasma cells exhibit dysregulated proliferation and depend on autophagy and mTOR signaling for survival. The well-characterized signaling background makes U266 an optimal host for gene knockout studies.

TMEM97 encodes the sigma-2 receptor, a key regulator of lysosomal cholesterol trafficking that interacts with PGRMC1, LDLR, and NPC1. Upstream regulators include SREBP1/2, mTORC1, and sigma receptor ligands, while downstream targets encompass mTORC1, NPC1, LC3B, SQSTM1/p62, and caspase-3. Knockout abolishes sigma-2 function, disrupting cholesterol export from lysosomes, impairing mTORC1 signaling and autophagy, and leading to lysosomal dysfunction, reduced proliferation, and enhanced apoptosis.

In multiple myeloma, TMEM97 loss exposes dependencies between cholesterol metabolism and oncogenic signaling. U266 cells rely on autophagy and mTORC1 for proteostatic control; thus, TMEM97 knockout allows dissection of how lysosomal cholesterol handling influences these survival pathways. The model supports evaluation of combination therapies with proteasome inhibitors or sigma-2-targeted agents.

Applications include co-immunoprecipitation of TMEM97 complexes, Western blotting for autophagy markers (LC3B, SQSTM1), and RT-qPCR analysis. Functional assays encompass cholesterol trafficking by Filipin staining, lysosomal pH measurement, flow cytometry for apoptosis (Annexin V/PI), and drug sensitivity profiling. Transcriptomic analysis via RNA-seq and LC3B puncta imaging further extend the utility. For additional information, contact Ascent Research.