Figure 4.

Sfrp2 promotes cardiomyocyte differentiation via of Wnt3a inhibition. (a) On day 3 of the differentiation protocol, cells were incubated with either WntC59 (2 μM) or Sfrp2 (1 nM) for 2 days. After two days, cell extracts were analyzed for total-β-catenin by immunoblotting. Total-β-catenin levels were normalized to the housekeeping protein GAPDH and are shown as a fold change compared to the control. Control protein extracts were isolated from cells prior to the addition of WntC59/Sfrp2. Representative immunoblots are shown. N = 3. Comparisons are made to control cells: ***P < 0.001. An image showing the immunoblot edge is shown in Supplementary Fig. 1. Note, unlike the images provided here, the image in Supplementary Fig. 1 is non-linear data. As such, it is unsuitable for quantification purposes. (b & c) On day 3 of the differentiation protocol, cells were incubated with a human Wnt3a neutralizing antibody (nAb) for 48 h. Cardiomyocyte differentiation was then assessed (day 14) by flow cytometry for cTnT. Representative flow traces are shown in (b) with quantification of the number of cardiomyocytes provided in (c). N = 4. Comparisons made to control cells (antibody vehicle): **P < 0.01. (d) On day 3 of the differentiation protocol, cells were incubated with WntC59 (2 μm) and the indicated doses of human Wnt3a protein for 48 h. Cardiomyocyte differentiation was assessed (day 14) by flow cytometry for cTnT and the fold change determined by normalizing to the control group (WntC59 treatment and Wnt3a protein vehicle). N = 4. Comparisons are made to the control group: ***P < 0.001. (e) On day 3 of the differentiation protocol, cells were incubated with human Wnt3a protein (100 ng/ml) and either vehicle or or Sfrp2 (1 nM) for 48 h. Cardiomyocyte differentiation was assessed (day 14) by flow cytometry for cTnT and the number of cardiomyocytes are expressed as a percentage of the total cell population. N = 3. Comparisons are made to the control group: **P < 0.01.