Fig. 2: Knockout of SLFN11 reprograms the G3PS through downregulation of mitochondrial GPD2.

a Heatmap showing relative mRNA expression levels from RNA-seq analysis for SLFN11, GPD2, GPD1, GPD1L, and AGPAT4 across WT and SLFN11^−/− EWS cell lines ES-8, SK ES-1, EW-8, RD ES-1, and CADO ES-1. Color scale represents relative gene expression. Each gene is independently scaled, and the accompanying color bar represents the relative expression range for that gene across EWS cell lines. b Volcano plot showing differentially expressed genes in ES-8 SLFN11^−/− versus ES-8 WT cell line from RNA-seq analysis. Red dots indicate significantly upregulated genes involved in G3PS (glycerol-3-phosphate shuttle), while blue dots represent significantly downregulated genes. c Western blot analysis of GPD2 (mitochondrial glycerol-3-phosphate dehydrogenase) and SLFN11 protein expression in ES-8 WT and SLFN11^−/−, SK ES-1 WT and SLFN11^−/−, and EW-8 WT and SLFN11^−/− cell lines. β-Actin serves as a loading control. d Bar plot showing GPD2 mRNA expression (log₂ [TPM + 1]) across 24 EWS cell lines from the DepMap dataset. Full-length uncropped Western blots corresponding to this figure are provided in the Supplemental Material. Correlation analysis between log₂-transformed SLFN11 and GPD2 read counts across EWS tumors in the e ESCLA (Ewing Sarcoma Cell Line Atlas) dataset (R = 0.51, p = 2.1e-08) and f DepMap dataset (R = 0.32, p = 1.92e-07). R represents the Pearson correlation coefficient. Statistical significance was defined as p < 0.05. g Schematic of the G3PS showing cytosolic conversion of DHAP (dihydroxyacetone phosphate) to G3P (glycerol-3-phosphate) via GPD1/1L (cytosolic glycerol-3-phosphate dehydrogenase), followed by mitochondrial oxidation of G3P by GPD2, transferring electrons to the ETC (electron transport chain) to support OXPHOS (oxidative phosphorylation). G3P also serves as the backbone for GPL (glycerophospholipid) biosynthesis in the cytosol.