Extended Data Figure 9: Quantitative analysis of NADPH consumption for biomass production and antioxidant defence.

a, Cell doubling times, which are inversely proportional to biomass production rates. b, Cellular protein content. c, Cellular fatty acid content (from saponification of total cellular lipid). d, Quantification of fatty acid synthesis versus import, with synthesis but not import requiring NADPH. HEK293T cells were cultured in [U-¹³C]glucose and [U-¹³C]glutamine until pseudo-steady state, and fatty acids saponified from total cellular lipids and their labelling patterns measured (green bars), and production versus import of each fatty acid was stimulated based on this experimental data. The fractional contribution of each route was determined by least square fitting, with the theoretical labelling pattern based on the elucidated routes shown (pink bars). Similar data were obtained also for MD-MBA-468, iBMK-parental, and iBMK-Akt cells (not shown) and used to calculate associated NADPH consumption by fatty acid synthesis. e, Cellular DNA and RNA contents. f, NADPH consumption by de novo DNA synthesis. g, Proline and glutamate labelling patterns after 24 h in [U-¹³C]glutamine media, which was used to quantitate different proline synthesis routes and associated NADPH consumption. h, Quantitative analysis of cytosolic NADPH consumption in normally growing HEK293T cells (control) and non-growing cell under oxidative stress (150 µM H₂O₂, 5 h). Total cytosolic NADPH turnover was measured based on the absolute oxidative pentose phosphate pathway flux divided by the fractional contribution of the oxidative pentose phosphate pathway to total NADPH as measured using [²H]NADPH formation from [1-²H]glucose. Mean ± s.d., n = 3.