Figure 3.

Exogenous Pyruvate Modulates TCA-Cycle Activity in Maintenance and Mesoderm Differentiation

(A) Schematic showing the glycolysis pathway and the functions of relevant inhibitors.

(B) qRT-PCR analysis of the impact of 3 mM 2-DG during BMP4-induced differentiation with or without 16 mM pyruvate treatment for 2 days (n = 3 independent experiments, ^∗p < 0.05).

(C) qRT-PCR analysis of the impact of 3 μM UK5099 during BMP4-induced differentiation with or without 16 mM pyruvate treatment for 2 days (n = 3 independent experiments, ^∗p < 0.05; ns, not significant).

(D) Schematic of pyruvate dehydrogenase (PDH) and the function of pyruvate and PDK inhibitor DCA.

(E) Western blotting analysis of PDHE1α phosphorylation during maintenance and differentiation, with or without 8 or 16 mM pyruvate treatment. Results shown are representative of three independent experiments.

(F) qRT-PCR analysis of the impact of 3 mM DCA during BMP4-induced differentiation with or without 16 mM pyruvate treatment for 2 days (n = 3 independent experiments, ^∗p < 0.05; ns, not significant).

(G) Levels of key metabolites during maintenance and differentiation, measured by LC-MS analysis. H1 cells were cultured with or without 16 mM pyruvate for 24 h with or without BMP4 induction in E8 medium. Metabolite levels were normalized to the levels in control cells without BMP4 or pyruvate treatment (n = 3 biological repeats, ^∗p < 0.05).

(H) Effects of metabolites on early mesoderm differentiation. Pyr, sodium pyruvate; α-KG, dimethyl 2-oxoglutarate; Suc, dimethyl succinate (n = 3 independent experiments, ^∗p < 0.05; ns, not significant compared with control). None, no pyruvate treatment.

See also Figure S3.