L 161 982

Conditioned medium of L-cell line secreting Wnt3a, R-spondin3, and Noggin (L-WRN CM) was prepared as described previously^{23 (link)}. L-WRN cells were purchased from ATCC (ATCC; CRL3276). Isolated crypts of the proximal colon were embedded in Matrigel (BD Biosciences). For spheroid culture of normal epithelium, 50% L-WRN CM supplemented with 5 µM Y-27632 (Tocris Bioscience) was added to each well. For EP4 antagonist experiment, spheroids generated from control (Villin-Cre) mice were treated with or without specific EP4-antagonist L161,982 (100 µM, Sigma) for 48 h and then analyzed.

The antibodies used in this article are listed in Table S1. The following reagents were also used: prostaglandin E2 (PGE₂; Cayman Chemicals, Ann Arbor, MI, USA, 14010), TGFβ (Peprotech, Cranbury, NJ, USA, 100‐21), IL4 and IFNγ (both from Immunotools GmbH, Friesoythe, Germany, 11340045 and 12343534), IL6 (Prospec, Rehovot, Israel, HZ‐1019), osteopontin (OPN, BioLegend, San Diego, CA, USA, 763602), TGFβ Receptor inhibitor SB505125 (S4696), prostaglandin EP2 Receptor inhibitor L‐161982 (SML‐0690), prostaglandin EP4 Receptor inhibitor PF‐04418948 (PZ0213), difluoromethylornithine (DFMO, D193‐25MG), celecoxib (70008, all five from Sigma‐Aldrich, Saint Louis, MO, USA), and the Stat3 inhibitor S3i‐201 (Selleckchem, Houston, TX, USA, S1155).

Rat primary ventricular neonatal cardiomyocytes (PVNC) were isolated from 1–2-day old pups using the Pierce Primary Cardiomyocyte Isolation Kit (#88281), which includes a cardiomyocyte growth supplement to reduce fibroblast contamination. H9c2 cells (ATCC CRL-1446) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone), containing penicillin, streptomycin, and 10% fetal bovine serum (Hyclone). Media was supplemented with MEM Non-Essential Amino Acids Solution (Gibco) for MEFs. Cells were incubated at 37 °C and 5% CO2. Human induced pluripotent stem cell-derived cardiomyocytes (H-iPSC-CMs) were obtained from Cellular Dynamics (iCell Cardiomyocytes #01434). iCell Cardiomyocytes were cultured in maintenance medium as per the manufacturer’s protocol and differentiated for 72 h. Cell lines were transfected using JetPrime Polyplus reagent, as per the manufacturer’s protocol. For misoprostol treatments, 10 mM misoprostol (Sigma) in phosphate buffered saline (PBS; Hyclone) was diluted to 10 μM directly in the media and applied to cells for 24 h. To achieve hypoxia, cells were held in a Biospherix incubator sub-chamber with 1% O₂ (±1%), 5% CO₂, balanced with pure N₂ (regulated by a Biospherix ProOx C21 sub-chamber controller) at 37 °C for 24 h. BvO2, L161-982, L798-106, and H89 dihydrochloride (H89) were purchased from Sigma.