Extended Data Figure 4: Probing the contribution of alternative NADPH producing pathways.

a, Pathway diagram showing potential for [2,3,3,4,4-²H]glutamine to label NADPH via glutamate dehydrogenase and via malic enzyme. Labelled hydrogens are shown in red. b, NADP⁺ and NADPH labelling patterns (without correction for natural ¹³C-abundance) after 48 h incubation with [2,3,3,4,4-²H]glutamine. The indistinguishable labelling of NADP⁺ and NADPH implies lack of NADPH redox active hydrogen labelling. c, Pathway diagram showing potential for [2,3,3-²H]aspartate to label NADPH via isocitrate dehydrogenase. d, NADP⁺ and NADPH labelling patterns (without correction for natural ¹³C-abundance) after 48 h incubation with [2,3,3-²H]aspartate. The indistinguishable labelling of NADP⁺ and NADPH implies lack of redox active hydrogen labelling. e, Diagram of [2,3,3,4,4-²H]glutamine metabolism through TCA cycle, tracing labelled hydrogen. Hydrogen atoms of lighter shade indicate potential H/D exchange with water. f, Malate labelling fraction after cells were supplied with [2,3,3,4,4-²H]glutamine for 48 h. g, Pathway diagram showing potential for [1,2,3-¹³C]malate (made by feeding [U-¹³C]glutamine) to label pyruvate and lactate via malic enzyme. h, Extent of malate and pyruvate/lactate ¹³C-labelling. Cells were incubated with [U-¹³C]glutamine for 48 h. M+3 pyruvate indicates malic enzyme flux, which may generate either NADH or NADPH. Similar results were obtained also for M+3 lactate, which was used as a surrogate for pyruvate in cases in which lactate was better detected. The corresponding maximal possible malic enzyme-driven NADPH production rate ranges, depending on the cell line, from < 2 nmol µl⁻¹ h⁻¹ (based on the limit of detection of M+3 pyruvate) to 6 nmol µl⁻¹ h⁻¹. Mean ± s.d., n ≥ 2.