Extended data Figure 3: Inhibiting lactate transporters down-regulates Wnt signaling.

(a) Whole-mount in situ hybridization of a 2-day chicken embryo hybridized with MCT1 (n=4). Scale bar: 100 μm.

(b) Comparison of lactate amounts in cellular extracts of the posterior region of 2-day chicken embryos cultured for 10 h in chemically defined medium with or without 5 mM CNCn (n=3). Mean+/− SD. Two-sided unpaired t-test. P=0.0292. *p<0.05.

(c) Whole-mount in situ hybridization of 2-day chicken embryos cultured with 0 (CTL) or 5 mM CNCn and hybridized with AXIN2 (control n=8, 5mM CNCn n=7). Scale bar: 100 μm.

(d) qPCR analysis of MSGN1, SAX1, SOX2, T and AXIN2 expression in the posterior region of 2-day chicken embryos cultured with or without 5 mM CNCn (n=3 for each gene). Data were normalized by control samples. Mean +/− SD. Two-sided unpaired t-test. AXIN2, p= 0.0197, T, p= 0.0270, SOX2, p= 0.0458. * p<0.05.

(e) Comparison of AXIN2 and SOX1 mRNA expression in day 2 human iPS cells differentiated in vitro and cultured for 24h in CL medium containing 5mM CNCn or vehicle control (DMSO). n=3 biological replicates. Mean +/− SD. Two-way ANOVA followed by Tukey’s multiple comparisons test: ***p= 0.0004; **p= 0.0067. n=3.

(f) Western blot analysis using anti-acetylated K49 β-catenin, anti-active β-catenin, anti-actin and anti-β-catenin antibodies of whole cell extracts of 2-day chicken embryos cultured in chemically defined medium with 0 or 5mM CNCn for 10h. (n=3).

For gel source data, see Supplementary Figure 1