Af2444

Western blotting was performed according to standard procedures using peroxidase-conjugated secondary antibodies and SuperSignal^® West Pico chemiluminescent substrate (Thermo). Primary antibodies were EOMES (Abcam #ab23345, 1:1000), GAPDH (Thermo #AM4300, 1:10,000), and NKX2.5 (R&D #AF2444, 1:100). All uncropped immunoblots can be found in Supplementary Fig. 5.

Cell numbers per well were quantified following single‐cell dissociation with Accutase and using standard Neubauer counting chambers. RT‐qPCR and immunocytochemistry were performed according to standard procedures. Primers validated for high‐efficiency amplification are given in Table S2. RPL37A served as housekeeping gene for normalization. Antibodies used were anti‐ISL1 (DSHB #39‐405, 1:15, and R&D Systems, Minneapolis, Minnesota, #AF1837, 1:100), anti‐CTNT (ThermoFisher Scientific #MS‐295‐P, 1:150), and anti‐NKX2.5 (R&D #AF2444, 1:100).

Immunofluorescence analysis was carried out according to standard procedures using secondary Alexa-488 or Alexa-568-conjugated antibodies and Hoechst for fluorescent staining. Primary antibodies were α-actinin (Sigma #A7811, 1:800), albumin (R&D #MAB1455, 1:100, albumin-free staining procedure), ANP (R&D #AF3366, 1:100), α-fetoprotein (Sigma A8452, 1:500), COL3A1 (Santa Cruz #sc-8780-R, 1:100), HA tag (Santa Cruz #sc-806-X, 1:200), MLC2v (ProteinTech Group #10906-1-AP, 1:200), NKX2.5 (R&D #AF2444, 1:100), PECAM1 (R&D #BBA7, 1:50), smooth muscle actin (DakoCytomation #M085129-2, 1:100), SOX2 (R&D #AF2018, 1:200), cardiac troponin I (cTnI, Santa Cruz #sc-15368, 1:200), cardiac troponin T (cTnT, Thermo #MS-295-P, 1:200), renin (R&D #AF4090, 1:150), β-III-tubulin (Covance #MMS-435P, 1:500), and vimentin (Sigma #V6630, 1:200).