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Fig. 5 | Stem Cell Research & Therapy

Fig. 5

From: Wnt/β-catenin-mediated signaling re-activates proliferation of matured cardiomyocytes

Fig. 5

Wnt signaling activated proliferation variations between treatments of antibody, small molecule, or both. a Representative images of untreated human ES cell-derived cardiomyocytes (control) and treated human ES cell-derived cardiomyocytes with either N-cadherin antibody (1:1000), CHIR99021 (1 μM), or combinations of both N-cadherin antibody and CHIR99021 (1:1000; 1 μM) stained for cTnT (green) and Ki67 (red). Matured human ES cell-derived cardiomyocytes were enriched with SIRPA 2 months after initial contraction. Note the increased in number of Ki67+/cTnT+ cardiomyocytes treated with N-cadherin antibody, CHIR99021, or combinations of N-cadherin antibody and CHIR99021 as compared to control. Cell nuclei (blue) were stained with DAPI. Scale bar, 100 μm. b, c Graphical representation of the proportion of Ki67+ cardiomyocytes in respective culture conditions. All treated cardiomyocytes (cTnT+) resulted in an increase in proliferating (Ki67+) cells as compared to control. Error bars indicate s.d, n = 3 experiments. *P < 0.05 and **P < 0.01 for Kruskal-Wallis one-way analysis of variance compared to control. d Representative FACS plots illustrating SIRPA+ cardiomyocytes upon culture in various conditions (control, N-cadherin antibody (1:1000), CHIR99021 (1 μM), and N-cadherin antibody (1:1000) + CHIR99021 (1 μM)) for 7 days. e Graphical representation showing absolute number of cardiomyocytes determined via FACS in the various culture conditions. Error bars indicate s.d., n = 3 replicates. **P < 0.01 for Kruskal-Wallis one-way analysis of variance compared to control. f Immunoblot showing increased expression of cytoplasmic β-catenin when matured cardiomyocytes were cultured in the presence of CHIR99021 (1 μM), N-cadherin antibody (1:1000), or both. Cell lysates were probed with antibodies against cytoplasmic β-catenin and β-actin (loading control). Densitometry of band intensities was performed using ImageJ software. g Immunostaining of matured human ES cell-derived cardiomyocytes with either anti-PHH3 or anti-Survivin antibody after treatment with CHIR99021 (red). The results revealed that more cells undergo cytokinesis upon treatment with CHIR99021 (1 μM) as compared to the control. Cardiomyocytes were stained for cTnT (green). White arrow shows cardiomyocytes that were either cTnT+/PHH3+ or cTnT+/Survivin+. Cell nuclei (blue) were stained with DAPI. Scale bar, 100 μm

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