Fig 2: Enrichment analysis of MTHFD2 functional networks in ovarian cancer. A Biological process. B Cellular component. C Molecular function. D KEGG pathway. E PANTHER pathway. F Reactome pathway

Fig 3: MTHFD2 expression provides survival advantage to GBM cells from glutamine deprivation. See also Additional File 2: Supplemental Fig. 4. a, b U87 cells were transfected with two types of MTHFD2 siRNA and scrambled control siRNA constructs for 24 h and changed to medium with or without glutamine at Day 1. Cell number over time represents the mean ± SEM of three independent experiments (statistically significant with **p < 0.01). Immunoblot images of MTHFD2 and actin were obtained from cell lysate. c Representative images of U87 cells with TUNEL staining. Scale bar: 100 μm. Cells were transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with or without glutamine for 48 h. Quantification of TUNEL-positive cells was performed with the ImageJ analysis. Data represent the mean ± SEM of three independent images for each group (statistically significant with **p < 0.01). d NAD +/NADH ratio in U87 cells transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with ± glutamine and ± NADH 1 mM for 48 h. Data represent the mean ± SEM of three independent experiments (statistically significant with **p < 0.01). e Representative fluorescence microscopy images of ROS signal in U87 cells. Scale bar: 100 μm. ROS measurement in U87 cells transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with ± glutamine for 48 h. ROS signal was inhibited by an antioxidant, 50 mM N-acetyl cysteine (NAC). Data represent the mean ± SEM of three independent experiments (statistically significant with *p < 0.05, **p < 0.01)

Fig 4: MTHFD2 expressions are elevated in the glutamine-deprived cells and tumor core of GBM patients. See also Additional File 2: Supplemental Fig. 3. a A schematic showing the enzymes involved in one-carbon metabolism that were targeted in this study. PSAT1; phosphoserine aminotransferase 1, SHMT1 and 2; serine hydroxymethyl transferase 1 and 2, and MTHFD1 and 2; methylenetetrahydrofolate dehydrogenase 1 and 2. MTHFD1L; monofunctional tetrahydrofolate synthase, mitochondrial b mRNA levels of PSAT1, SHMT1 and 2, MTHFD1 and 2, and MTHFD1L in U87 and T98 GBM cells which were grown with or without glutamine for 48 h. Data represent the mean ± SEM of three independent experiments (statistically significant with *p < 0.05, **p < 0.01). c Immunoblot analysis of PSAT1, SHMT1 and 2, MTHFD1 and 2 staining in central tumors (T) and normal brain tissues (N) around tumor edge obtained at tumor resection from 6 patients with GBM. d Representative immunohistochemical images of MTHFD2 in central tumors obtained from a GBM patient. Tissue was counterstained with hematoxylin. Scale bar upper 200 μm, lower 100 μm arrow; pseudopalisading asterisk; necrosis

Fig 5: Co-Expression of MTHFD2 gene. A Co-expression of MTHFD2 gene as detected by cBioPortal. B Regression analysis between MTHFD2 and MOB1A in ovarian cancer determined by cBioPortal. C Relationship between MTHFD2 and MOB1A in ovarian cancer by GEPIA. D Heat map of MTHFD2 and MOB1A mRNA expression in ovarian cancer identified by UCSC Xena. E Correlation between MTHFD2 and MOB1A mRNA expression in the TCGA database, identified by UCSC Xena