Fh535

Prostaglandin E2 was from Sigma (St. Louis, MO). Sulprostone (Sulp) was from ABCAM (Cambridge, UK). Butaprost (Buta) was from Santa Cruz Biotechnology (Dallas, USA). Prostaglandin E1 Alcohol (POH), L161982, AZD5363, FH535, and ICI 182780 (ICI) were from Cayman Chemical (Detroit, USA). Antibody of prostaglandin E synthase 2 (Anti-PTGES2) was from Proteintech (Chicago, USA). Antibodies of prostaglandin E receptor 4 (Anti-EP4), SUMO-1, and SUMO-2, 3 were from ABCAM (Cambridge, UK). ELISA Kit for Prostaglandin E2 (PGE2) was from Cloud-Clone Corp (Houston, USA).

Primary SFZ cells were isolated as previously described [21 (link)]. Briefly, the primary end of the femur and the distal end of the tibia were dissected from P5 mice, and ligaments and tendons were excised. Cartilage tissues were incubated with 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA) for 1 h, followed by 1.5-h digestion with 173 U/mL of type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ). DZ cells were isolated by additional digestion of residual epiphyseal cartilage tissue with 43 U/ml collagenase type I for 5 h. Dissociated cells were seeded on fibronectin-coated culture dishes. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS). The cells were cultured as a monolayer in all experiments. We used 2 μM 4-hydroxytamoxifen (4OHT; Sigma Aldrich, St Louis, MO, USA) to induce Cre recombination in the cultured SFZ cells. Recombinant human (rh) WNT5A (R&D Systems, Minneapolis, MN), rhWNT5B (R&D Systems), rhWNT9A (R&D Systems), CHIR99021 (Cayman, Ann Arbor, MI), FH535 (Cayman), and 666-15(Tocris Bioscience, Bristol, UK) were used at the concentrations described in the figures and figure legends. For the luciferase assay, ATDC5 cells (Riken BRC, Tsukuba, Japan) were cultured in DMEM/F-12 (Wako) containing 5% FBS.