Extended Data Figure 5: Computational and experimental evidence for THF-dependent NADPH production.

a, Predicted contribution of folate metabolism to NADPH production based on flux balance analysis, using minimization of total flux as the objective function, across different biomass compositions. The biomass fraction of cell dry weight consisting of protein, nucleic acid and lipid was varied as follows: protein 50–90% with a step size of 10%; RNA/DNA 3–20% with step size of 1%, and lipids 3–20% with step size of 1% (considering only those combinations that sum to no more than 100%). With this range of physiologically possible biomass compositions, the model predicts a median contribution of folate metabolism of 24%. Note that with the constraint of experimentally measured biomass composition, yet without constraining the uptake rate of amino acids other than glutamine to be ≤ 1/3 of the glutamine uptake rate, the contribution of folate pathway to total NADPH production is predicted to be 23%. b, Range of feasible flux through NADPH producing reactions in Recon1 model computed via flux variability analysis under the constraint of maximal growth rate. As shown, the model predicts that each NADPH producing reaction can theoretically have zero flux, with all NADPH production proceeding through alternative pathways. Only reactions whose flux upper bound is greater than zero are shown. Reactions producing NADPH via a thermodynamically infeasible futile cycle were manually removed. As shown, among all NADPH producing reactions, MTHFD has the highest flux consistent with maximal growth. c, Pathway diagram showing potential for [2,3,3-²H]serine to label NADPH via methylene tetrahydrofolate dehydrogenase. d, NADP⁺ and NADPH labelling pattern after 48 h incubation with [2,3,3-²H]serine (no glycine present in the media). The greater abundance of more heavily labelled forms of NADPH relative to NADP⁺ indicates redox active hydrogen labelling. Results are mean ± s.d., n ≥ 2 biological replicates from a single experiment and were confirmed in n ≥ 2 experiments. Based on the data in panel d, the contribution of MTHFD1 to cytosolic NADPH production spans a broad range (10–40% of total cytosolic NADPH; the range is due to variation across cell lines, experimental noise, and the large KIE⁴⁰). This range includes the flux calculated based on purine biosynthetic rate and ¹⁴CO₂ release from serine (Fig. 3d). Note that the total contribution of the cytosolic folate metabolism to NADPH production can exceed that of MTHFD1, as 10-formyl-THF dehydrogenase also produces NADPH.