Extended Data Fig. 1. Data supporting Fig. 1.

a, PI3K-regulated metabolite screen. Serum/growth factor-deprived (18 h) MCF10A cells treated (1 h) with insulin (100 nM) and PI3K inhibitor (GDC-0941, 2 μM). Graphed metabolites were significantly (one-way ANOVA, p<alpha 0.05) different by ≥ 50% between any two treatments in both independent experiments (A and B). b, Diagram of heavy isotope (¹³C₃¹⁵N₁)-labelled Vitamin B5 (VB5) tracing into CoA and acyl-CoAs. Labelled carbon (blue), nitrogen (red). Labelled VB5, CoA, and acyl-CoAs are all native mass + 4 [M+4]. c, Steady state ¹³C₃¹⁵N₁-VB5 labelling of MCF10A cells (4–6 days) in media containing only ¹³C₃¹⁵N₁-VB5, supplemented with either defined serum replacement or horse serum. Metabolites measured by LC-MS/MS and displayed as fractional abundance of labelled metabolites ((M+4)/total). Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. d, Growth of MCF10A cells over 4 days in serum-free media containing growth factors and defined serum replacement, in the presence or absence of 1 µM VB5. Cells were grown in the absence of VB5 for 48 h prior to plating. Mean (points on line); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-sided student’s t-test, (p<alpha 0.05). e, Time course of MCF10A cells serum/growth factor-deprived (18 h), pre-incubated (1 h) with ¹³C₃¹⁵N₁-VB5, and treated (1, 3, or 5 h) with insulin (100 nM) and ¹³C₃¹⁵N₁-VB5 labelling. Protein immunoblots probed with antibodies to total and phosphorylated (p) AKT (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-way ANOVA with Sidak’s test, (p<alpha 0.05). f, Validation of MS/MS methods for ¹³C₃¹⁵N₁-VB5 tracing into VB5, CoA, and acetyl-CoA. Serum/growth factor-deprived (18 h) MCF10A cells were treated (3 h) with insulin (100 nM) and labelled with ¹³C₃¹⁵N₁-VB5. Unlabelled fructose-1,6-bisphosphate (F-1,6-BP) shown as control. Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). g, Time course showing intracellular levels of ¹³C₃¹⁵N₁-VB5 in serum/growth factor deprived (18 h) MCF10A cells upon incubation in media exclusively containing ¹³C₃¹⁵N₁-VB5 for 10–60 min. Graphed metabolites measured my LC-MS/MS. Mean (points on line); SEM (error bars); n = 3 biological replicates.