a, Mammalian iBMK cells cultured on [5-2H1]- or [1,2-13C2]glucose after addition of DMSO (untreated) or oligomycin for 30 min. On oligomycin treatment, the glycolytic intermediates retained more 2H and the unlabeled DHAP pool decreased, indicating less reversibility. The center and error bars represent the mean ± s.e.m. (n = 3, biologically independent samples). b, Corresponding ΔG were inferred from the flux-modeling results that best fit the observed 2H and 13C labeling. Each of the reaction(s) ΔG (<0) is represented by the height of the green bar. The bottom edges indicate the cumulative ΔG up to the corresponding step in glycolysis. Whiskers show s.e.m. (Methods). On oligomycin treatment, glycolysis shifted forward, towards a stronger thermodynamic force, especially at the aldolase and GAPD:PGK steps. *P < 0.05 and **P < 0.01 by two-tailed t-tests or bootstrapping (Methods).