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. 2013 Jan 22;31(5):928–940. doi: 10.1002/stem.1332

Figure 6.

Figure 6

Inhibition of β-catenin-dependent transcription rescues cardiac differentiation in FHL2-ESC-EBs. (A): Schematic representation of in vitro cardiogenesis in embryonic stem cell (ESC)-EBs upon 5 μmol/l QC application from day 9 or 11 of differentiation. Cells were harvested at day 16 of differentiation for analysis. (B): Transcriptional effect of quercetin treatment on differentiating FHL2-ESCs. qPCR analysis showed unchanged β-catenin expression but reduced levels of upregulated Tcf4 and Igfbp5 expression in FHL2-ESC-EBs upon quercetin treatment in comparison to untreated FHL2-ESC-EBs following 16 days of differentiation. mRNA levels were normalized to Gapdh. (C): Blocking β-catenin transcriptional upregulation via quercetin increased the expression of cardiomyocyte genes in FHL2-ESC-EBs. Quercetin treatment at day 11 reversed the decreased αMHC-GFP expression during cardiac differentiation in FHL2-ESC-EBs (triangles and discontinues red lines) in comparison to untreated FHL2-ESC-EBs (dots and continues red lines) expressed as percentages of total EBs. Quercetin did not affect expression of αMHC-GFP in wild-type-ESC-EBs (black lines). Representative confocal immunofluorescence pictures of the αMHC-GFP expression are shown (right). (D): Flow cytometry shows a rescue in upregulation of NKX2.5 as well as in downregulation of α-MHC-GFP/α-sarcomeric-actinin-expressing cells in FHL2-ESC-EBs compared to untreated FHL2-ESC-EBs at 16 days of differentiation. Data represent mean ± SEM; (B): n = 6, (C): n = 10, (D): n = 3, ***, p < .001 (ANOVA; Bonferroni's multiple comparison test). Scale bar = 100 μm. Abbreviations: EB, embryoid body; FHL2, four-and-a-half-LIM-domain 2; GFP, green fluorescent protein; QC, quercetin.