3 isobutyl 1 methyl xanthine (ibmx)

For osteogenic differentiation, the cells (2.5 × 10⁴ cells/well in 24-well plates) were cultured in an osteogenic medium containing a growth medium supplemented with 250 nM dexamethasone (Cat. no. D8893, Sigma-Aldrich, USA), 50 µg/mL ascorbic acid (Cat. no. A-4034, Sigma-Aldrich, USA), and 5 mM β-glycerophosphate (Cat. no. G9422, Sigma-Aldrich, USA) for 14 days. Alkaline phosphatase enzymatic activity (ALP) and mineralization were examined using ALP staining and alizarin red S (ARS) staining, respectively (n = 4).
For the adipogenic differentiation, the cells (1.25 × 10⁴ cells/well) were maintained in 24-well plates with adipogenic medium comprising a growth medium containing 1 µM dexamethasone (Cat. no. D8893, Sigma-Aldrich, USA), 0.1 mg/mL insulin (Cat. no. 11070738 Sigma-Aldrich, USA), 0.2 mM indomethacin (Cat. no. 53861, Sigma-Aldrich, USA), and 1 mM IBMX (Cat. no. PHZ1124, Thermo Fisher Scientific, USA) for 16 days. The intracellular lipid droplet accumulation was examined using oil red o staining (n = 4).

cAMP levels were assessed using the AlphaScreen Detection Kit (Perkin Elmer, Waltham, MA, USA). All cells were serum-starved for at least three hrs prior to the analysis. Briefly, cells were resuspended at 5 × 10⁵ cells/ml in AlphaScreen stimulation buffer containing HBSS (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.5 mM IBMX, 5 mM HEPES, 0.1% BSA (all from Thermo Fisher Scientific, Waltham, MA, USA), pH 7.4 in the presence or absence of forskolin (10 μM final concentration) (Sigma Aldrich, St. Louis, MO, USA) and treated or untreated with A/C heterodimerizer (100 mM final concentration). 3000 cells/well were aliquoted in triplicate into 384-well OptiPlate (Perkin Elmer, Waltham, MA, USA) and stimulated with CXCL12 and CCL19 chemokine ligands at indicated concentrations for 15 min at 37 °C. Further, cells were processed according to the manufacturer’s protocol. Alpha Screen signal was recorded using PHERAstar® FSX detection system (BMG Labtech, Ortenberg, Germany) using the AlphaScreen optical module (Ex. 680 nm/Em. 520−620 nm). Data analysis was performed using GraphPrism software (San Diego, CA, USA).

The cells were seeded into 24-well plates (Thermo Scientific) at a density of $2\times {10}^4$ cells/cm ${}^2$ and cultured for 16–20 days with an adipogenic medium containing 10% FBS-DMEM supplemented with 500 $\mathsf{\mu }$ mol 3-isobutyl-1-methylxanthine (IBMX) (cat. No. PH21124, Thermo Scientific), 1 $\mathsf{\mu }$ g/mL insulin from bovine pancreas, 100 $\mathsf{\mu }$ M indomethacin (cat. No. I7378, Sigma-Aldrich) that was kept at room temperature before use. The medium was changed every 2 days.

Riociguat (Selleck Chemicals), tadalafil (Sigma-Aldrich/Supelco), DMSO, forskolin (Sigma-Aldrich), vinpocetine, EHNA (Enzo Life Sciences), BAY60-7550, TAK-063 (Cayman Chemical), milrinone, vardenafil, zaprinast (Santa Cruz Biotechnology), sildenafil (Merck), IBMX (Thermo Fisher Scientific) and DEA/NO (Axxora).

hFPTs and ASC-F cells were seeded at a relative viable density of 1.5 × 10³ cells/cm² in multiple 12-well cell culture microplates (Falcon^®, USA) in CM-FBS or CM-HPL growth medium, respectively. Cultures were appropriately maintained in both incubation conditions (i.e., normoxia and hypoxia) until cell monolayers attained 80% confluency (i.e., 6 ± 2 days). Thereafter, the specific culture medium was replaced with a specific adipogenic induction medium, composed of high-glucose DMEM (Gibco™, USA) supplemented with 10% v/v FBS (Sigma-Aldrich^®, USA), 2 mM L-glutamine (Gibco™, USA), ITS 1 × (i.e., final concentrations of 10 mg/L insulin, 5.5 mg/L transferrin, and 6.7 µg/L selenious acid, Corning^®, USA), 1 µM dexamethasone (Acros Organics™, ThermoFischer Scientific, USA), 100 µM indomethacin (Acros Organics™, USA), and 100 µM IBMX (Alfa Aesar™, ThermoFisher Scientific, USA). The induction medium was exchanged twice weekly for a period of 14 days for the different cell types and in both incubation conditions. At the end of the induction period, the cells were fixed with a 4% formalin solution and appropriately stained with Oil Red-O (Sigma-Aldrich^®, USA) for revelation of lipid droplet accumulation. Following staining, assay microplates were photographed on an Olympus CX30 contrast phase microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).