Extended Data Fig. 1: Mitochondrial pyruvate transport and amino acid metabolism influence spheroid growth.

a, HCT116 spheroid growth from 3-, 5- and 8-day cultures. Scale bars, 100 μm. b, Metabolite levels in HCT116 adherent and spheroid cultures (n = 3 culture wells each). c, Alanine levels in adherent and spheroid cultures (n = 3 culture wells each condition and cell line). d, Isotopic labelling (M2 citrate/M3 pyruvate) in HCT116 and MCF7 cells cultured with [U-¹³C₆]glucose for 24 h (n = 3 culture wells each). e, Protein expression of phosphorylated PDH (pPDH), total PDH (tPDH) and β-actin in HCT116 cells. Each lane derived from a single culture well, processed in parallel, and used for quantification. For gel source data, see Supplementary Information. f, Citrate labelling in HCT116 cells cultured with [U-¹³C₅]glutamine in HCT116 (n = 3 culture wells each condition). g, Metabolite levels upon UK5099 treatment in HCT116 spheroid cultures (n = 6 culture wells each). h, Abundances of alanine and serine in A549 spheroid cultures upon treatment with UK5099 (n = 3 culture wells each). i, Spheroid growth in cells upon UK5099 treatment (n = 3 culture wells each). j, k, Adherent growth of A549 (j) and HCT116 (k) cells upon treatment with UK5099 (n = 3 culture wells each). l, m, Adherent growth of A549 (l) and HCT116 (m) cells upon MPC1 or MPC2 knockdown compared to control (shNT) (n = 3 culture wells each). n, o, Isotopologue distributions of serine (n) and citrate (o) in HCT116 spheroid cultures traced with [U-¹³C₆]glucose for 24 h (n = 3 culture wells each). p, Alanine abundances in HCT116 spheroids in the presence of 1 mM alanine and UK5099 (n = 3 culture wells each). q, r, Spheroid growth of HCT116 (q) and MCF7 (r) cells grown in the presence of UK5099 and alanine (n = 3 culture wells each condition). s, Cell number of adherent HCT116 cells in the presence of UK5099 and alanine (n = 3 culture wells each). t–v, Spheroid biomass in HCT116 (t), MCF7 (u) and A549 (v) cells grown in the presence or absence of 0.4 mM serine, 0.4 mM glycine, 1 mM alanine and 1 mM formate (n = 3 culture wells for each cell line and condition). Two-sided Student’s t-test (b–i, n, o), one-way ANOVA (t–v) or two-way ANOVA (j–m, p–s) was performed for each comparison, with no adjustment for multiple comparison. Similar results obtained in two (d, e, h, p, r), three (c, f, g, i, n, o, q), or four (b) independent experiments. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.0001.