Fig. 8: Rnf20-dependent EC-derived factors regulate CM proliferation.
a Schematic representation of the NicheNet analysis of significant ligand–receptor pairs considering differentially expressed ligands in ECs and receptors on CMs in Isl1cre/+Rnf20fl/fl and Rnf20iEC-KO versus control embryos. The dot plots depict ligands contributing to the CM response in Rnf20iEC-KO vs control embryos. Cardiac cell type schematics created in BioRender.com. Dobreva, G. (2025) https://BioRender.com/ce62pb8. b Heatmap representation of the expression of Rnf20 and TGF-β members/ downstream targets in CHD patients. c Schematic representation of the co-culture experiment design involving rCMs and transfected HUVECs. Transwell co-culture system schematics created in BioRender.com. Dobreva, G. (2025) https://BioRender.com/yr6530z. d Immunostaining for EdU and MF20 (left panels) and percentage of proliferating EdU-positive CMs in co-cultures with HUVECs transfected with control siRNA (n = 3), siRNA against RNF20 (siRNF20, n = 3), siRNF20 supplemented with recombinant NRG1 (n = 3) or IGF2 (n = 3), or siRNF20 together with siRNAs against various collagens (n = 3) or SMAD4 (n = 3), determined by EdU-incorporation for 24 h (right panel). Scale bars, 50 µm. e Immunostaining for EdU and MF20 (left panels) and percentage of proliferating EdU-positive CMs in co-cultures with HUVECs transfected with control siRNA (n = 3), siRNA against RNF20 (siRNF20, n = 3), or siRNF20 together with siRNAs against SMAD2 and SMAD3 (n = 3), determined by EdU-incorporation for 24 h (right panel) (n = 3 per group). Scale bars, 50 µm. Statistical analysis between multiple groups in (d, e), was performed using one-way-ANOVA with Šídák’s correction. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
