Extended Data Figure 9: CoQ10 synthesis is important for growth in 3D, but not 2D, conditions.

a, Schematic of pulse-chase study using tyrosine-derived intermediates shown in c. Cells were labelled with ¹³C₉-Tyr for two weeks before being grown in ¹²C₉-Tyr or ¹³C₉-Tyr with or without unlabelled 4-HPPA, 4-HPLA, 4-HMA, and 4-HB for 24 h at 21% O₂. b, Growth curve of MIAPACA2 cells with the indicated intermediates and times at 21% O₂. (n = 3 technical replicates for each cell line, performed at least twice). c, Total levels and fractional labelling of unlabelled and ¹³C-labelled metabolites in the CoQ10 headgroup biosynthesis pathway in humans, as described in a (n = 4). Endogenous 4-HB is below the limit of detection. d, Schematic of known and potential enzymes and intermediates in the CoQ10 headgroup biosynthesis pathway in humans and yeast. Dotted lines reflect potential pathways and enzymes. e, Immunoblot of the indicated MIAPACA2 cells. ERK2 is the loading control. Experiment was performed twice to check for knockout efficiency. f, Growth in 2D culture of MIAPACA2 cells (n = 4). g, h, Total levels (g) and fractional labelling (h) of CoQ10 in MIAPACA2 cells. Cells were grown in ¹³C₉-Tyr at 21% ¹⁶O₂ for 24 h (n = 5). i, Growth in 3D culture of MIAPACA2 cells (n = 4) after three days. j, Representative confocal fluorescent images of the indicated MIAPACA2 cells. Images are representative of three independent experiments. k, Fractional labelling of intracellular and mitochondrial CoQ10 from ¹³C₉-Tyr in the indicated MIAPACA2 cells (n = 4). l, Immunoblot of total, cytosolic, and mitochondrial fractions from k. Subcellular fractionation was performed twice to determine the localization of HPDL. n represents the number of biologically independent experiments for each group and condition, unless indicated (b). Graphs (mean ± s.e.m.) were compared by two-way ANOVA (b, c, f–i, k), followed by Tukey (b, c, f–h, k) or Dunnett’s (i) post hoc test (*P < 0.05, ^P < 0.01, ^%P < 0.005, ^#P < 0.0001).

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