FBXO31-mediated Ubiquitination of OGT Maintains O-GlcNAcylation Homeostasis to Restrain Endometrial Malignancy

CRISPR screen and biochemical characterization reveal that tumor suppressor F-box only protein 31 (FBXO31) regulates O-GlcNAcylation homeostasis in EC by ubiquitinating the O-GlcNAc transferase OGT. Overall design: The human GeCKO v2 library was transduced into 293T cells by lentivirus at a multiplicity of infection of 0.3. Cells were selected with puromycin for 7 days followed by fluorescence-activated cell sorting (FACS) based on their O-GlcNAcylation staining intensities. An unsorted sample was used to assess sgRNA library coverage, and the sorted RL2 high population was subjected to genomic DNA extraction. The inserted sgRNA library was amplified by two steps of PCR for next-generation sequencing. Each screen was performed twice.