Isobutyl 1 methylxanthin

Experiments were performed as previously reported (Wu et al., 2021). To measure intracellular cyclic adenosine monophosphate (cAMP) concentration changes, cells were cultivated in a 96-well plate until 90% confluence. Aldehyde 13-13 was diluted in induction buffer, consisting of Ringer’s solution, 500 μM isobutyl-1-methylxanthin, and 100 μM 4-imidazolidin-2-one (Sigma-Aldrich). After incubation for 30 min, intracellular cAMP concentrations were measured using cAMP Glo Assay Kit (Promega) and Fusion α Multi-well Plate Reader (Packard Bioscience, Wellesley, MA, USA). Data were normalized to the solvent control (ethanol). Treatment for 30 min with 10 μM forskolin (Sigma-Aldrich) served as the positive control.

HFDPC 3D cultures were induced 24 h after cell encapsulation. Cultures were maintained under adipogenic and osteogenic medium for 21 days. Control medium consisted of DMEM supplemented with 10% FBS, L-Glutamine and P/S. Osteogenic medium consisted on control medium supplemented with 50 µM L-ascorbic acid 2-phosphate (A8960, Sigma-Aldrich), 100 nM Dexamethasone (D8893, Sigma-Aldrich) and 10 mM β-Glycerol-2-phosphate (G9422, Sigma-Aldrich) [17 (link)]. Adipogenic medium consisted on control medium supplemented with 10 µg/mL insulin (I9278, Sigma), 100 nM Dexamethasone (D8893, Sigma-Aldrich), 0.2 mM Indomethacin (I7378, Sigma-Aldrich) and 0.5 mM isobutyl-1-methylxanthin (I7018, Sigma-Aldrich) [17 (link)].