Kaluza 1.1 acquisition software

EMT stage was evaluated by analyzing the protein expression of the selected epicardial markers ALDH1A1, TCF21 and WT1. ACM and HC-CMSC were harvested, fixed and permeabilized using the Fixation/Permeabilization kit (BD Biosciences, Franklin Lakes, NJ, USA). CMSCs were incubated with the proper primary and secondary antibodies (Table S5): ALDH1A1 (Abcam), TCF21 (Abcam), WT1 (Abcam); Goat anti-Rabbit 488 (Invitrogen). Fluorescence detection for 5000 events per sample occurred with the Gallios flow cytometer platform by using Kaluza 1.1 acquisition software, and the analysis was conducted using Kaluza 1.3 analysis software (Beckman Coulter).

The proliferation rate of CMSCs was tested using an EdU assay kit (Abcam), an index of DNA replication. CMSCs were seeded with a density of 2.5 × 10⁴/cm² cells, and after 24 h, they were incubated with medium, supplemented with 40 μM EdU, at 37 °C for 4 h. Subsequently, CMSCs were detached, fixed with the fixative solution (4% formaldehyde-based) and permeabilized with the permeabilization buffer (Triton X-100-based). For EdU detection, cells were incubated with iFluor 488 azide and measured with the Gallios flow cytometer platform by using Kaluza 1.1 acquisition software (Beckman Coulter, Brea, CA, USA). Data from 5000 events per sample were collected and examined with Kaluza 1.3 analysis software.