Camp xp assay kit

For cAMP assays, A549 human lung epithelial cells were suspended in Ham's F‐12K (Thermo Fisher Scientific Inc.) containing 10% heat‐inactivated FBS and seeded on 24 well plates as 50,000 cells/500 μL/well for 24 h. Cells were treated with Compound 1 (1 × 10^‐10 mol/L to 1 × 10^‐5 mol/L) dissolved in HBSS (Thermo Fisher Scientific Inc.) containing 0.1% bovine serum albumin (BSA) and 5 mmol/L HEPES for 30 min and stimulated with 10 μmol/L forskolin and 10 μmol/L prostaglandin E2 for 30 min. Cells were lysed and cAMP content in cell lysates and supernatants were measured using cAMP XP Assay kit (Cell signaling technology, MA).

Roflumilast (5 mg/kg) and Compound 1 (10 mg/kg) were administered to C57BL/6N mice (Charles River Laboratories Japan, Inc., Japan) which were then sacrificed at 0.5, 1, 2, 4, 8, and 24 h after the dosing. Blood samples were collected and centrifuged to obtain plasma specimens. Plasma concentrations of roflumilast, the active metabolite of roflumilast N‐oxide and Compound 1 were measured using liquid chromatography/tandem mass spectrometry. Bronchoalveolar Lavage Fluid (BALF) was collected by instilling three aliquots of 0.5 mL 0.9% saline into the lungs. BALF was centrifuged to remove cells, the supernatant was deproteinized with trichloroacetic acid, and washed by diethylether. The aqueous layer was freeze‐dried and dissolved by lysis buffer enclosed in cAMP XP Assay kit (Cell Signaling Technology Inc., Danvers, MA). cAMP content was measured in accordance with the manufacturer's instruction, and, total PDE4 inhibitory activities (tPDE4i) were calculated by following equation: $\text{tPDE4i}=\sum \frac{(C_n\text{or}{\text{AUC}}_n)\times {\text{fu}}_n}{{\text{IC50}}_n}$ where C_n and AUCn represent the plasma concentration and the AUC of active pharmacological ingredient (API), respectively, fu is the unbound fraction in plasma, IC50 is the concentration resulting in 50% inhibition in an in vitro assay and n represents the number of APIs (Hermann et al. 2007). Of note, roflumilast has two APIs and Compound 1 has one API.

Semi-confluent cells plated in 24-well plates were treated in triplicate for 30 min with forskolin (0, 0.1, 0.3, 1, or 3 μM) (Calbiochem, Merck, Damstadt, Germany) plus rikkunshito stock solution (0, 1, or 3%). Cells were then subjected to a competition enzyme-linked immunoassay by using the cAMP XP™ assay kit (Cell Signaling Technology, Danvers, MA).

TM4 and TM4-FSHR cells were seeded (0.3 × 10⁶ cells/plate) and cultured in 6 cm plates. The next day, after starvation for 4 h, the cells were stimulated with recombinant FSH (Follitrope, LG Chem, Seoul, South Korea) or SAFA-FSH at different concentrations in 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37°C. The reaction was stopped by aspirating the medium and washing twice with cold Dulbecco’s phosphate-buffered saline (DPBS). Cells were lysed with 80 μL of lysis buffer and incubated on ice for 30 min. Cell lysates were obtained by centrifugation at 14000 ×g for 10 min at 4°C. cAMP concentrations were measured using a cAMP XP assay kit (Cell Signaling Technology, Danvers, MA, USA). In each experiment, a standard curve was generated and used to calculate the cAMP concentration.