Figure 4

Combination of XMU-MP-1 and S1P increases cardiac myocyte proliferation. (A) Heat map showing percentage of BrdU-positive nuclei in a preliminary screen of combinatorial treatment of hiCMs with XMU-MP-1 and S1P. N = 8393 cells from 5 independent experiments. (B) BrdU+ datapoints from black outlined diagonal center boxes from heat map. N = 2229 cells from 5 independent experiments. (C) Proliferation of control hiCMs or 0.1 µM XMU-MP-1 and 0.1 µM S1P-treated cells measured by counting nuclei over 48 h. N = 17,318 DMSO-treated cells and 16,234 XS-treated cells from 4 independent experiments. Significance determined by two-tailed paired Student’s t test. (D) Western blot of hiCMs treated with various concentrations of both XMU-MP-1 and S1P. pYAP: phosphorylated YAP (see Figure S1F,G for the entire blot). (E) pYAP amount in hiCMs measured by western blot normalized to tubulin (N = 3 independent experiments). (F) The compounds XMU-MP-1, S1P, and Verteporfin do not affect the percentage of beating hiCMs at the doses given (1 µM for XMU-MP-1 and S1P, 0.1 µM for XMU-MP-1 and S1P combined, and 10 nM Verteporfin. In last row, 0.1 µM of XMU-MP-1 and S1P were combined with 10 nM Verteporfin). (G) The compounds XMU-MP-1 and S1P do not affect the percentage of binucleated and mononucleated hiCMs at the same doses as (F). (H) Schematic of YAP’s downstream transcriptional regulation and interaction with TEAD. (I) Verteporfin dose response to detect which concentration of Verteporfin inhibited hiCM proliferation, but did not lead to massive cell death. (J) Proliferation of control hiCMs compared to 0.01 µM Verteporfin, or 0.01 µM Verteporfin, 0.1 µM S1P, and 0.1 µM XMU-MP-1-treated hiCMs. N = 1257 cells from 4 independent experiments. (K) Proliferation of control hiCMs compared to 0.01 µM Verteporfin, or 0.01 µM Verteporfin and 0.1 µM S1P, and 0.1 µM XMU-MP-1-treated hiCMs. Significance determined by one-way ANOVA with a Dunnett’s post-hoc test correcting for multiple testing for panels (B), (E), (F), (G), and (J).