Comprehensive Metabolic Flux Analysis using INCA

¹³C MFA was conducted using INCA, a software package based on the EMU framework (http://mfa.vueinnovations.com; ref. 29 (link)). Intracellular concentrations of free metabolites and intra- and extracellular fluxes were assumed to be constant over the course of the tracing experiment. Fluxes through a metabolic network comprising of glycolysis, the pentose phosphate pathway, the TCA cycle, biomass synthesis, and fatty acid synthesis were estimated by minimizing the sum of squared residuals between experimental and simulated MIDs and extracellular fluxes using nonlinear least squares regression (30 (link)). The best global fit was found after estimating 100 times using random initial guesses for all fiuxes in the network. A χ² statistical test was applied to assess the goodness-of-fit using α of 0.01. The 95% confidence intervals for all fluxes in the network were estimated by evaluating the sensitivity of the sum of squared residuals to flux variations (30 (link)). Isotopomer Spectral Analysis was performed as previously described (31 (link)). See Supplementary Methods for further details of MFA.