Fig. 4: SLFN11 knockout increases GPL biosynthesis in EWS.

a MSEA-based pathway enrichment analysis identifying upregulated metabolic pathways in ES-8 SLFN11^−/− cell line. Pathways with –log₁₀(FDR) ≥ 0.5 were considered enriched and ranked by significance. Asterisks mark statistically significant pathways. b Correlation analysis of log₂-transformed SLFN11 and AGPAT4 expression across EWS tumors using patient-derived microarray data [25] from the Gene Expression Omnibus (GEO) database. (R = −0.38, p = 2.3e-04). R represents the Pearson correlation coefficient. Statistical significance was defined as p < 0.05. c Schematic illustrating showing U-¹³C acetate incorporation into the GPL biosynthesis pathway. Cyan circles represent ¹³C-labeled acetate-derived carbons incorporated into fatty acid chains. Gray circles denote the synthesis of G3P backbone. Enzymes involved in GPL biosynthesis are highlighted in maroon. Isotopologue distribution of U-¹³C acetate-labeled GPL species, PE (34:1) d, PC (34:1) e, PG (34:1) f, and PI (34:1) g in ES-8 WT and SLFN11^−/− cells. Bars represent the relative abundance of individual mass isotopologues (M + 2 to M + 22), corresponding to successive incorporation of ¹³C-labeled acetate units during GPL synthesis. A color scale denoting isotopologue species (M + 0 to M + 22) is included alongside the figure. Data represent mean ± SD (n = 5). Statistical comparisons were performed using paired two-tailed Student’s t test for each comparison and the corresponding p-values are reported in Table S9. The asterisk denotes statistical significance for the M₀ isotopologue (p < 0.05). SD, standard deviation; n, number of replicates. h Schematic of U-¹³C acetate tracing into de novo fatty acid synthesis. Cyan circles represent ¹³C-labeled acetate-derived carbons incorporated during elongation. The diagram illustrates labeling flow into saturated and monounsaturated fatty acid species. Distribution of U-¹³C acetate-labeled isotopologues in palmitate (i) and oleate (j) in ES-8 WT and SLFN11^−/− cells. Color code for isotopologues (M + 0 to M + 22) is shown adjacent to panel. Bars represent mean ± SD (n = 5). Statistical comparisons were performed using paired two-tailed Student’s t test for each comparison. SD standard deviation; n number of replicates.