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. 2020 May 23;10(15):6898–6914. doi: 10.7150/thno.42347

Figure 4.

Figure 4

MESP1 represses canonical Wnt/β-CATENIN signaling during hESC cardiac differentiation. (A) Dox inducible MESP1 expression system. Left, dosage effect of Dox on MESP1-Flag protein expression (Dox concentration is indicated at the top). Middle and right, MESP1-Flag protein was induced 3 h after Dox addition and disappeared 6 h after Dox withdraw. ACTIN was used as the loading control in all experiments. (B) Schematic view of the differentiation protocol. Cells were treated with 25 ng/mL BMP4 and 2 µM CHIR99021 for 24 h. 48 h after BMP4 and CHIR99021 removal, 5 µM IWP2 or 400 ng/mL Dox was added for 48 h. After IWP2 or Dox treatment, cells were cultured in RPMI/B27 minus insulin medium until day 7, then changed to RPMI/B27 medium until day 12. (C) Bar graph showing cTnT+ cell percentage in untreated, Dox, or/and IWP2 treated cells based on flow cytometry analysis (n = 3). Data represent mean ± SEM. (D) Q-PCR analysis of genes induced by MESP1. Dox or IWP2 were added during day 3-5. The expression levels of cardiac and Wnt pathway genes were normalized against GAPDH. The expression levels in untreated cells were set as “1”. (n = 3). (E) Western blot showing MESP1, phosphorylated β-CATENIN (phos-β-CATENIN), and total β-CATENIN protein levels in cells treated IWP2 or Dox, ACTIN was used as the loading control (n = 3). (F) Schematic view of WNT5A promoter region. Dark blue boxes indicated E-box motifs. Red lines indicate regions tested in ChIP-Q-PCR assay. TSS: transcription start site. (G) Timeline of ChIP-Q-PCR analysis. (H) ChIP-Q-PCR analysis of MESP1 binding to E-box motifs on the WNT5A promoter. A non-related genomic region was used as negative control. Results were normalized against the enrichment of the same DNA fragment without Dox induction. A, B, C and D are E-box regions depicted in (F). (I) Luciferase reporter assay showing the transcription activity of E-box A (WNT5A promoter); mut E-box A (WNT5A promoter with E-box A mutated); ΔE-box A (E-box A deleted WNT5A promoter). Empty vector was used as negative control. Data represent the mean ± SEM, n = 3, *p < 0.05, **p < 0.005, ***p < 0.001, Student's t-test. (J) Western blot showing induction of WNT5A in hESCs reduced the level of total β-CATENIN protein. GAPDH, loading control. (K) Schematic view of differentiation protocol, cells were treated with WNT5A instead of IWP2 during day 3-5. (L) FACS analysis of cTnT on day 12 of differentiation.