Fig. 2: Key CTLH E3 ubiquitin ligase roles in EBVaGC survival and metabolism regulation.
a Schematic of CTLH, adapted from Mohamed et al.28,57. Screen hits are in red. b Immunoblot of WCL from YCCEL1 expressing control vs. MAEA sgRNA. c Mean ± SD live cell ratios from n = 3 replicates of YCCEL1 in (b) treated with 0.5 μM alpelisib vs DMSO. P-values by two-tailed one-way ANOVA. d Representative n = 3 biologically independent replicate bright-field images of (c). e Cell cycle analysis of propidium iodide (PI) stained YCCEL1 in (c). Based on FACS analyses (left), percentages of cells (right) in cell cycle phases were calculated. Representative n = 3 biologically independent replicates. f MAEA cDNA rescue. Left, immunoblot analysis of WCL from YCCEL1 expressing GFP vs CRISPR-resistant MAEA cDNA with control or MAEA sgRNA. Right, mean ± SD live cell ratios from n = 3 replicates of YCCEL1 expressing control GFP vs MAEA cDNA and treated with 0.5 µM alpelisib for 7 days. P-values by two-tailed one-way ANOVA. Live cell values were normalized to DMSO-treated cells. g, h Metabolic pathway impact analysis from liquid chromatography mass spectrometry (LC/MS) analyses (n = 6 independent replicates) of YCCEL1 expressing MAEA vs control sgRNAs in (g) DMSO and (h) 0.5 μM alpelisib for 30 h. x-axis shows pathway impact values, y-axis shows the -log10 P-value from MetaboAnalyst 3.0109 Topology analysis. i Glycolysis heatmap. Row Z-scores from LC/MS analysis of YCCEL1 cells in (g) and (h). Z-scores show SDs from the mean value in each row. j 2-NBDG uptake in YCCEL1 expressing Control (Ctrl) or MAEA sgRNAs and treated with DMSO or alpelisib (ALP). Means ± SD mean fluorescence intensity (MFI) from n = 3 independent replicates. P-values are analyzed by two-tailed two-way ANOVA. k Oxygen consumption rates (OCRs) of YCCEL1 as in (g) and (h) following addition of oligomycin A, CCCP, and antimycin. Mean ± SEM from n = 6 independent replicates. l Basal OCR of YCCEL1 as in (k). Mean ± SD from n = 6 independent replicates. P-values by two-tailed one-way ANOVA. Immunoblot analysis is representative of at least n = 3 biologically independent replicates. Source data are provided as a Source Data file for (b), (c), (e), (f), and (j–l).
