Ultrav block

For flow cytometry analyses, cells were trypsinized, resuspended as single cells, permeabilized in 0.1% Triton X-100 at 4 °C for 10 minutes, blocked with UltraV block (Fisher Scientific) at room temperature for 7 minutes, incubated with primary anti-Vimentin, anti-cTnT, and anti-cardiac alpha sarcomeric actinin (cASA) antibodies (Table) or isotype control antibodies at 4 °C overnight, washed, and stained with appropriate secondary antibodies (Table). After staining, the cells were washed, re-suspended in 2% FBS/PBS containing 5 μL of propidium iodide (10 μg/mL), and evaluated with a FACS Aria instrument (BD Biosciences).
For immunohistochemistry analyses, hciPSCs and hciPSC-CMs were fixed with 4% paraformaldehyde at room temperature for 20 minutes, permeabilized in 0.1 Triton X-100 at 4 °C for 10 minutes, and blocked with UltraV block for 7 minutes. Primary antibodies against SSEA4, Tra1-60, and Oct4 (hciPSCs), or cTnT and connexin43 (hciPSC-CMs) were added to the UltraV block buffer at a concentration of 1:100, and the cells were incubated at 4 °C overnight; then, the labeled cells were washed and incubated with FITC-,and TRITC-conjugated secondary antibodies (Table) in UltraV block buffer at room temperature for 1 hour, counterstained with DAPI, washed, and visualized under a fluorescence microscope (Olympus, Japan).

Differentiated hiPSC-ECs were fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized in 0.1% Triton X-100 at 4°C for 10 minutes, and then blocked with UltraV block (Fisher Scientific, USA) for 7 minutes. Primary antibodies (monoclonal anti-CD31 and mouse anti-CD144 [BD Pharmingen, USA]; goat anti-vWF [Santa Cruz Biotechnology, USA]) at a concentration of 1:100 were added to the UltraV block buffer and incubated overnight at 4°C; then, the cells were incubated with FITC-or TRITC-conjugated secondary antibodies (donkey anti-mouse or goat IgG) in UltraV block buffer for 1 hour at room temperature, labeled with DAPI, washed, and viewed under a fluorescence microscope (Olympus, Japan).

Laboratory chemicals were from Sigma-Aldrich (Vienna, Austria) unless specified. The EP1 receptor agonist ONO DI-004, the EP4 receptor agonist ONO AE1-329 and the EP4 receptor antagonist ONO AE3-208 were kind gifts from ONO Pharmaceuticals (Osaka, Japan). PGE₂, 17-pt-PGE₂, SC51089, GW627368X, L-161,982, ONO-8711, iloprost, and isobutylmethylxanthine were from Cayman Chemical (Ann Arbor, MI, USA). L-161,798 was purchased from Tocris Biosciences (Bristol, UK) and SC51322 from Biomol (Hamburg, Germany). The VE-cadherin mouse monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and secondary fluorescently-labeled antibodies and Texas Red-X Phalloidin were purchased from Invitrogen (Invitrogen, Lofer, Austria). Antibody diluent was from Dako (Glostrup, Denmark), Ultra V Block from Fisher Scientific (Vienna, Austria). Vectashield/DAPI mounting medium was obtained from Vector Laboratories (Vector Laboratories, Burlingam, CA, USA).