3 isobutyl 1 methyl xanthine (ibmx)

Measurement of ligand-induced cAMP generation in GPR10 WT stably expressing cells was done using HitHunter® cAMP assay (DiscoverX, 90-0075SM) according to manufacturer’s protocol with modifications. 20,000 cells/well were seeded in a white poly-D-lysine coated 96-well plate and cultured overnight. The following day, cells were washed with PBS and incubated in 30 μL PBS supplemented with 1 mM 3-­isobutyl-1-methylxanthine (IBMX, Cayman Chemical, 13347) for 30 min prior to stimulation with an agonist. Cells were stimulated with serial dilutions of PrRP-31 (1.5 μL/well, 20x dilution, Bachem, 4028740) for further 30 min at 37 °C. Intracellular cAMP detection was carried out directly after the ligand stimulation. 10 μL anti-cAMP antibody followed by 40 μL chemiluminescent substrate/lysis buffer/enzyme donor-cAMP complex mix prepared according to manufacturer’s protocol were added and plates were incubated shaking for 1 h at ambient temperature. Finally, 40 μL enzyme acceptor was dispensed and chemiluminescent signal was quantified after 4-5 h in a TopCount 9012 Microplate Counter (Packard).

DMEM, OptiMEM, penicillin-streptomycin, PBS and sodium pyruvate were obtained from Gibco by Life Technologies (Carlsbad, CA, United States), SYBR green MicroAmp Fast Optical 96-well Reaction Plate 0.1 ml and StepOnePlus thermocycler were obtained from Applied Biosystems (Foster City, CA, United States). FCS was purchased from Capricorn (Ebsdorfergrund, Germany). New born calf serum was purchased from Biochrom (Berlin, Germany). All primer pairs were purchased from Eurofins (Friedrichsdorf, Germany). Cell culture flasks (T75, Cell+, vented cap) and 6-well plates for sub-culturing 3T3-L1 cells were obtained from SARSTEDT (Nümbrecht, Germany). Rosiglitazone, pioglitazone, zafirlukast, montelukast, IBMX and (±)14(15)-EET-d₁₁ were purchased from Cayman Chemical (Ann Arbor, MI, United States). Insulin, dexamethasone and Oil Red O were obtained from Sigma Aldrich (St. Louis, Missouri, MO, United States). Tris, Triton-X-100, NP-40, NaCl, EDTA and SDS were purchased from AppliChem (Darmstadt, Germany).

The C2C12BRELuc reporter cell line [41 (link)] was generated by the Inman group and used in this study. In this cell line, the luciferase (Luc) reporter is under the control of the BMP response elements (BRE) of the Id1 gene. C2C12BRELuc were cultivated in culture medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, all from Biochrom AG, Berlin, Germany). C2C12BRELuc cells were passaged twice per week in a 175 or 225 cm² cell culture flask. The cultivation media was supplemented with the antibiotic G418 (0.7 mg/mL) to select for the BREluc-positive C2C12BRELuc cells in accordance with previous studies [41 (link)].
C2C1BRELuc cells were sub-cultivated at a ratio of 2.5–5 × 10⁴ cells per mL of culture medium. DMEM, antibiotics, FBS, and trypsin (Biochrom AG, Berlin, Germany) were used in cell cultivation and passaging. To ensure the viability and to calculate cell number, the metabolic activity of cells was calculated through Presto Blue^® assay (Invitrogen by Life Technologies Co., Carlsbad, CA, USA). BMP2 was purchased from Osteogenetics (Würzburg, Germany). H89 was purchased from AbCam (Cambridge, UK). Deta NONOate, SNAP, YC-1, LY83583, LDN-193189, and IBMX were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Forskolin, IBMX, bafilomycin and epoxomicin were purchased from Cayman, Leupeptin from US Biological, cycloheximide from Calbiochem, SAG (Smoothened AGonist) from Enzo and CellTracker Deep Red from Thermo.

Genipin was purchased from BOC Sciences (Shirley, NY, USA) with purity > 98%; U73122 was from Tocris Bioscience (Pittsburgh, PA, USA); Dulbecco’s Modified Eagle Medium (DMEM) was from Hyclone (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA); fetal bovine serum (FBS) was from GenClone (Genesee Scientific, EL Cajon, CA, USA); penicillin–streptomycin was from Sigma (St. Louis, MO, USA); poly-D-lysine was from MP Biomedicals (Solon, OH, USA); trypsin-EDTA and GLP-1 ELISA kits for in vitro experiments were from MilliporeSigma (Burlington, MA, USA); Fluo-4AM was from ThermoFisher Scientific (Waltham, MA); bovine serum albumin (BSA), diprotin A, methylcellulose, IBMX, cyclic AMP ELISA kit, alanine aminotransferase (ALT) kit, and triglycerides colorimetric assay kit were from Cayman (Ann Arbor, MI, USA); leptin assay kit was from Bertin Pharma (Montigny-le-Bretonneux, France); total GLP-1 ELISA kits for plasma assay were from Crystal Chem (Elk Grove Village, IL, USA); CellTiter-Blue^® cell viability assay reagent was from Promega (Madison, WI, USA); glucose meter and strips were from AgaMatrix (Salem, NH, USA); and all other chemicals were from Sigma (St. Louis, MO, USA).