Fig. 5: Screen for TSGs that regulate O-GlcNAcylation homeostasis.

a Schematic representation of the FACS-based genome-wide CRISPR-Cas9 screen for putative regulators of O-GlcNAcylation homeostasis. Fluorescence-Activated Cell Sorting (FACS); Immunofluorescence (IF); CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; Human genome-wide CRISPR/Cas9 knockout (GeCKO). The elements in this figure were created using BioGDP.com (https://BioGDP.com). b Validation of the sensitivity of RL2 staining (red) with 293T cells transfected with OGT (green). Nuclei are labeled with DAPI (blue). Scale bar: 5 µm. c Genes plotted according to their relative ranking analysis (RRA) enrichment scores, with known O-GlcNAcylation regulators highlighted in red and blue. Knockdown (KD). d Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showing enrichment of putative O-GlcNAcylation regulators in the indicated pathways (statistical analysis was performed using a hypergeometric test to calculate p-values). Analysis was performed on the 1038 high-confidence genes (p < 0.05). e Venn diagram showing the overlap between the 526 human UCEC Tumor Suppressor Genes (TSGs) and the 1038 high-confidence genes from the O-GlcNAcylation screen. Source data are provided in the Source Data file. f Immunofluorescent detection of O-GlcNAcylation level by RL2 (red) in WT (Wild Type) and FBXO31-KO (FBXO31-Knockout) 293T cells. Nuclei were stained with DAPI (blue). Scale bar: 5 µm. g Kaplan-Meier analysis of the OS of the EC patients stratified by the expression level of FBXO31 (http://kmplot.com/analysis/). EC cases were stratified using the median cut-off, and statistical significance was determined using the log-rank test. Results in (b, f) show a representative example from n = 3 independent experiments.