Fig. 6

Tregs secrete a pool of proteins promoting CM proliferation. a Supernatants from CHO cells transfected with plasmids encoding for the factors listed on the X-axis of the graph were added to rat neonatal CMs and proliferation scored as EdU incorporation. All the tested proteins were included in Pool12, whereas only those exerting a pro-proliferative effect individually were included in Pool6. Black bars correspond to proteins included in Pool6, whereas gray bars correspond to proteins included in Pool12 but not in Pool6. The supernatant of cells transfected with an empty plasmid (SN pGi) was used as a negative control. At least 500 cells were analyzed per supernatant in each replicate using the MetaXpress software. b Representative pictures showing EdU incorporation by rat neonatal CMs exposed to the supernatant of CHO cells transfected with the negative control pGi, Pool6, and Pool12. Lower panels show higher magnification of the are defined by the white dotted line in the upper panels. Nuclei are stained blue with DAPI and CMs green by anti-α-actinin antibodies. Red nuclei indicate EdU incorporation. Scale bar, 25 μm. c Representative images of whole transverse sections after Azan Trichromic staining of hearts injected with an empty AAV vector (Control) or AAV vectors expressing the proteins included in Pool6 and Pool12 at 15 and 60 days after myocardial infarction. Fibrotic areas are stained in gray/blue. Scale bar, 1 mm. d US imaging analysis of the left ventricular ejection fraction in animals injected with a control vector (C) and vectors expressing the proteins included in Pool6 and Pool12 at 1, 4, and 8 weeks (W) after permanent coronary artery ligation. e US imaging analysis of the left ventricular fractional shortening in animals injected with a control vector (C) and vectors expressing the proteins included in Pool6 and Pool12 at 1, 4, and 8 weeks (W) after permanent coronary artery ligation. f Sections of the heart of mice injected with a control vector, AAV-Pool6, and AAV-Pool12, showing EdU incorporation (stained red) in the nuclei of CMs (stained green). Nuclei are stained blue with DAPI. Scale bar, 50 μm. All values are mean ± s.e.m., each dot indicates a biological replicate. One-way analysis of variance and Bonferroni/Dunn’s post hoc tests were used to compare multiple groups in a. Two-way ANOVA for repeated measurements was used in (d). *P < 0.05, **P < 0.01, relative to control