Fig 2: MTHFD2 drives cancer cell proliferation independently of its enzymatic activity.(a–c) HCT-116 MTFHD2, HCT-116 MTHFD2ΔNAD or HCT-116 GFP cell lines cultured in medium containing 5% serum were treated +/− 1 μg/ml doxycycline and CyQuant assays of proliferation were carried out at indicated time points. Error bars are standard deviations. *P < 0.05. MTHFD2 expression was assayed by immunoblot analysis, β-Actin, ACTB, served as loading control, the images in the figure are cropped. Numbers indicate fold induction of MTHFD2 upon DOX treatment. (d) Light microscopy images of conditions as in (a) at time-point 96 hrs. (e,f) HCT-116 MTFHD2 or HCT-116 MTHFD2ΔNAD cell lines were treated +/− 1 μg/ml doxycycline for 48 hours to induce MTHFD2 or MTHFD2ΔNAD expression and then serum starved in 0.2% serum. CyQuant assays of proliferation were carried out at indicated time points. Error bars are standard deviations. *P < 0.05.

Fig 3: Association of higher expression of MTHFD2 with worse prognosis and malignant status of lung cancer. a Kaplan–Meier survival curves of patients in the NCC cohort. Red and blue lines depict survival curves of patients with cancer tissues with high and low levels of MTHFD2 mRNA, respectively. The cutoff value was determined as the median value of the expression levels of MTHFD2 mRNA. The p-values were calculated with a log-rank test. b Box-whisker plot of MTHFD2 mRNA for patients in different stages (edges of boxes represent the 25th and 75th percentiles and bold lines in boxes represent the median value). Whiskers were elongated to the largest and smallest values, which are not outliers. c Immunohistochemistry for MTHFD2 in normal and lung cancer tissues. Scale bar, 100 µm. d Comparison of MTHFD2 expression levels among lung cancer tissues that show different differentiation grades on the tissue microarray. Blue circles indicate scores of each patient. Statistical significance is calculated with Steel–Dwass method. N.S. not significant; *p < 0.05; **p < 0.01

Fig 4: Reduction in folate-mediated 1C metabolism and purine synthesis following MTHFD2 knockdown. a Intercellular concentrations of glycine (Gly) and serine (Ser). NCT negative control; shMTHFD2#53, MTHFD2 knockdown. The data are represented as mean ± SD, N = 3. b Ratio of the amount of NADH to NAD+ (NADH/NAD+) normalized to the value in control cells. The data are represented as mean ± SD, N = 3. c Concentrations of metabolites involved in nucleotide synthesis pathway. Deep blue and red bars depict concentrations of metabolites in negative control and shMTHFD2#53 cells, respectively. Dashed lines indicate multiple reactions. Reactions transferring 1C are depicted by red lines. The data are represented as mean ± SD, N = 3. d Concentrations of purine and pyrimidine nucleotides with each base A, G, C, and U. The data are represented as mean ± SD, N = 3. e Cell proliferation assay in the presence of hypoxanthine or sodium formate. Experiments were performed three times and the representative results were presented. The data are represented as mean ± SD, N = 3. Statistical significance is calculated with a two-tailed unpaired t-test; N.S. not significant, *p < 0.05, **p < 0.01

Fig 5: 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)