Extended Data Figure 2: Nutrient metabolism in H460 spheroid culture.

a, Cell proliferation and doubling times of H460 cells cultured under monolayer and spheroid conditions (n = 4 cultures days 1–4; n = 3 cultures days 5–6 from a representative experiment). b, Effect of glucose or glutamine deprivation on cell counts in monolayer and spheroid culture (n = 4 cultures from a representative experiment). c, Rates of glucose consumption and lactate excretion in monolayer and spheroid culture (n = 4 cultures from two experiments). d, Rates of glutamine consumption; glutamate and ammonia excretion; ratio of ammonia excretion to glutamine consumption; and rate of excretion of ¹⁵NH₄⁺ originating from [γ-¹⁵N]glutamine in monolayer and spheroid culture (n = 3 cultures from a representative experiment). e, Citrate mass isotopologue analysis in H460 cells in monolayer culture, aggregated into spheroids (P1), or disaggregated from spheroids then permitted to re-aggregate (P2) (n = 2 cultures from a representative experiment). f, Right, protein levels of phosphorylated PDHα (pPDH, Ser293), total PDHα (tPDH) and PDK1 in monolayer and spheroid culture with or without 2 mM dichloroacetate (DCA). Left, citrate mass isotopologue analysis in H460 spheroids cultured with [U-¹³C]glucose or [U-¹³C]glutamine, and treated with 2 mM DCA (n = 3 cultures from a representative experiment). All data represent mean ± s.d. *P < 0.05, Welch’s unequal variances t-test (a, c, d and f), or Welch’s unequal variances t-test, followed by multiple-comparison correction (b). All experiments were repeated 3 times or more.

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