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. 2019 Jul 25;13(2):338–351. doi: 10.1016/j.stemcr.2019.06.003

Figure 2.

Figure 2

Exogenous Pyruvate Potentiates Mesoderm Differentiation under BMP4 Induction through WNT Pathway

(A) Effect of pyruvate on BMP4-induced mesoderm differentiation. H1 cells were induced to mesoderm cell fate by BMP4 in E8 medium with or without pyruvate supplement and collected on day 2 for qRT-PCR analysis (n = 3 independent experiments, ∗p < 0.05).

(B) Dose response to pyruvate during mesoderm differentiation. H1 cells were induced toward mesoderm cell fate by BMP4 in the presence of pyruvate at different concentrations, and the expression of TBXT and MIXL1 was measured by qRT-PCR after 2 days. Data shown are normalized to day-0 levels (n = 3 independent experiments, ∗p < 0.05).

(C) FACS analysis of TBXT expression on day 3 of BMP4-induced differentiation with or without pyruvate treatment. Left: representative image of histogram. Right: bar graph showing mean ± SD of three independent experiments (∗p < 0.05).

(D) Immunostaining of TBXT on day 3 of BMP4-induced differentiation with or without pyruvate treatment. Control, no BMP4 treatment. Scale bars, 50 μm.

(E) Microarray analysis of gene expression in major lineages after 2 days and 6 days of pyruvate treatment during H1 maintenance or mesoderm induction.

(F) Time course of gene expression (TBXT/MIXL1) under pyruvate treatment during mesoderm differentiation, measured by qRT-PCR. Results are representative of three independent experiments.

(G) qRT-PCR analysis of WNT pathway-related genes during BMP4-induced differentiation with or without 16 mM pyruvate treatment for 2 days (n = 3 independent experiments, ∗p < 0.05).

(H) Schematic showing pyruvate modulation of BMP4- and WNT pathway-induced mesoderm differentiation. CHIR99021 is an activator of WNT pathway, and IWP2 is an inhibitor of WNT pathway.

(I) qRT-PCR analysis of the effect of WNT inhibitor IWP2 (3 μM) on mesoderm differentiation with or without pyruvate (Pyr) treatment for 2 days (n = 3 independent experiments, ∗p < 0.05).

(J) qRT-PCR analysis of the impact of pyruvate on 5 μM CHIR99021-induced mesoderm differentiation for 1 day (n = 3 independent experiments, not significant [ns]).

See also Figures S1 and S2.