Fig. 4. Onset of fibrotic phenotype. a. Representative immunofluorescence pictures of Ki67 (red). Nuclei were stained with DAPI (blue). Scale bars 100 μm. b. Quantification of total proliferating cells: quantification of αSMA positive cells over the total cellular population, quantification of myofibroblasts over the total fibroblast population and quantification of hypertrophic cardiomyocytes over the total number of cardiomyocytes. At least n = 5 images belonging to N = 3 biologically independent samples for each condition were used for quantifications. The percentage of αSMA positive cells was determined as the number of cells expressing αSMA over the total number of cells. CMs were defined as cTROP+ cells; αSMA expressing CMs as cTROP+– αSMA+ cells; myofibroblasts were defined as cTROP–αSMA+ cells; FBs were defined as cTROP–αSMA-cells. Statistics by one-way ANOVA with Tukey's post hoc test for Gaussian populations and Kruskal–Wallis test with Dunn's post hoc test for non-Gaussian populations. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. c. Representative images of α-SMA (cyan) and cardiac troponin I (green) (c) of the four considered conditions after 5 days of culture under dynamic conditions. Nuclei were stained with DAPI (blue). Scale bars 100 μm.