Extended Data Figure 2: Two independent measurement methods give consistent oxidative pentose phosphate pathway fluxes.

a, Diagram of [1-¹⁴C]glucose and [6-¹⁴C]glucose metabolism through glycolysis and the oxidative pentose phosphate pathway. The oxidative pentose phosphate pathway specifically releases glucose C1 as CO₂, whereas all other CO₂-releasing reactions are downstream of triose phosphate isomerase (TPI). As TPI renders C1 and C6 of glucose indistinguishable (both positions become C3 of glyceraldehyde-3-phosphate), the difference in CO₂ release from C1 versus C6, multiplied by two, gives the absolute rate of NADPH production via oxidative pentose phosphate pathway. A potential complication involves carbon scrambling via the reactions of the non-oxidative pentose phosphate pathway, but this was negligible (see Extended Data Fig. 3). b, Complete carbon labelling of glucose-6-phosphate. Glucose-6-phosphate was labelled completely (> 99%) within 2 h of switching cells into [U-¹³C]glucose. c, CO₂ release rate from [1-¹⁴C]glucose and [6-¹⁴C]glucose. d, Pool size of 6-phosphogluconate. e, Kinetics of glucose-6-phosphate and 6-phosphogluconate labelling upon switching cells to [U-¹³C]glucose. f, Overlay upon the 6-phosphogluconate data from e of simulated labelling curves based on the flux that best fits the labelling kinetics (blue) (see Methods), and the flux from ¹⁴CO₂ release measurements (green). g, Calculated fluxes and 95% confidence intervals based on kinetics of 6-phosphogluconate labelling from [U-¹³C-]glucose, compared to radioactive CO₂ release from [1-¹⁴C]glucose and [6-¹⁴C]glucose. The two approaches give consistent results, with the ¹⁴CO₂ release data being more precise. Mean ± s.d., n = 3.