3 isobutyl 1 methylxantine

Forskolin (FSK), H-89 dihydrocloride (H89), 3-isobutyl-1-methylxantine (IBMX), dimethyl sulfoxide (DMSO), 1,6-Hexanediol, Phosphate Buffered Saline (PBS), Tween 20, Bovine Serum Albumin (BSA) and Skim Milk Powder were from (Merck KGaA, Darmstadt, Germany). 8- (4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, acetoxymethyl ester (8-pCPT-2′-O-Me-cAMP-AM) was from BioLog (Biolog Life Science Institute GmbH & Co. KG, Bremen, Germany). SUMOylation Inhibitor III, 2-D08 was from Calbiochem. Adenosine 3′,5′-cyclic monophosphate (cAMP) for in vitro experiments was from Sigma-Aldrich (A6885).

To compare the multipotency of FBS-cultured with hPL-cultured hADSC, cells were differentiated into adipocytes and osteocytes. For both differentiation protocols, about 5000 cells (P3) were seeded in 12-well plates and maintained in differentiation medium for 2 weeks with the medium changed every 2 days. Adipogenic differentiation medium was composed of complete medium added with 1 μM dexamethasone (Sigma Aldrich), 100 μM indomethacin (Sigma Aldrich), 100 μM 3-isobutyl-1-methylxantine (Sigma Aldrich), and 1x ITS (Sigma Aldrich). The osteogenic differentiation medium was composed of complete medium added with 50 μg/mL ascorbic acid (Sigma Aldrich), 5 mM β-glycerophosphate (Sigma Aldrich), and 100 nM dexamethasone (Sigma Aldrich). Cells cultured with the corresponding complete medium without differentiation factors were used as a negative control. After the differentiation period, hADSC were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with Oil Red O (0.18%, Sigma Aldrich) for fat droplets evaluation or Alizarin Red (0.5%, Sigma Aldrich) to detect calcium deposits. Images were acquired with an optical microscope in bright field (IX81, Olympus, Iowa, USA). Experiments (adipogenic) were conducted in technical and biological triplicates, while the osteogenic one in three technical repeats and five biological repeats (5 donors)).

For adipocyte-progenitor-cell isolation, SV cells from subcutaneous adipose tissue from lean C57BL/6 mice were washed with PBS containing 0.5% BSA (Sigma-Aldrich), incubated with Fc block (Bioscience) and stained with anti-CD31 (FITC, Biolegend) for 20 min at 4 °C. CD31-positive cells were removed by using MACS magnetic beads (anti-FITC, Miltenyi). Afterward, the SV cells underwent CD45 positive selection to remove hematopoietic cells using MACS magnetic beads (anti-CD45, Miltenyi). Adipocyte progenitors were sorted as CD31⁻CD45⁻Sca1⁺ cells. The cells were resuspended in DMEM/F12 medium supplemented with 10% FBS. Then they were seeded into culture dishes until they reached 70–80% confluence.
For the differentiation of adipocyte progenitors into adipocytes, cells were seeded into 6-well plates at a density of 5 × 10⁴ cells per well in 2 ml complete DMEM/F12. When the cells reached confluence (day 0), cell medium was replaced with medium containing insulin (0.5 µg/ml, Sigma-Aldrich), 3-isobutyl-1-methylxantine (0.5 mM, Sigma-Aldrich), dexamethasone (1 µM), pioglitazone (1 µM, Sigma-Aldrich). After 48 h, medium was exchanged with complete DMEM/F12 containing insulin (0.5 µg/ml) and pioglitazone (1 µM). Differentiated adipocytes (days 8–10) were used for assays.

NKA and the peptide analogs of NKA listed in Table 1 were synthesized by Genscript (Piscataway, USA) to a purity ≤95%. NKA and substance P were purchased from Sigma Aldrich, (St. Louis, USA); septide was purchased from Bachem (Bubendorf, Switzerland, EU). [³H]-Septide was custom synthesized from Quotient Bioresearch (Cardiff, UK). [¹²⁵I]-NKA, and Microscint 20, were obtained from Perkin Elmer (Boston, USA). F12K medium, Geneticin (G418) and Lipofectamine 2000 were obtained from LifeTechnologies (Carlsbad, USA). Phosphate buffered saline (PBS) was from Lonza (Walkersville, MD, USA) and EDTA from Life Technologies (New York, USA). FuGENE HD Transfection Reagent was obtained from Promega (Madison, USA). The following reagents were obtained from Sigma Aldrich (St. Louis, USA): DMSO, Pluronic F-127, HEPES, bacitracin, SIGMAFAST Protease Inhibitor Tablets, bovine serum albumin (BSA), polyethileneimine (PEI), NaCl, 3-isobutyl-1-methylxantine (IBMX), and probenecid. The CRE/CREB Reporter Assay Kit, Firefly Luciferase reagent, and Renilla Luciferase reagent were purchased from BPS Bioscience (San Diego, USA).

The differentiation of MSCs in vitro towards the adipogenic and the osteogenic lineages were assayed as previously described [17 (link), 18 (link)]. Briefly, for adipocyte differentiation, MSCs were cultured for 3 weeks with adipogenic medium, containing 10⁻⁶ M dexamethasone, 10 μg/ml insulin and 100 μg/ml 3-isobutyl-1-methylxantine (Sigma, USA); for osteoblast differentiation, MSCs was cultured for 3 weeks with osteogenic medium, containing 10⁻⁷M dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (Sigma, USA). Oil-red-O and von kossa dyes were respectively employed to identify adipocytes and osteoblasts.

3T3-L1 cells used in the study were purchased from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/ penicillin (growth medium, GM) at 37 °C in the humid incubator with 5% CO₂. To induce differentiation, postconfluent preadipocytes were treated with hormonal inducers DMI [1 μg ml⁻¹ INS (Roche Diagnostics GmbH, Mannheim, Germany), 0.5 mM 3-iso-butyl-1-methylxantine (Sigma), and 1 μM dexamethasone (DEX) (Sigma) in DMEM containing 10% FBS]. Three days later, 3T3-L1 cells were cultured in DMEM supplemented with 10% FBS and 1 μg ml⁻¹ INS for 2 days. Then, 3T3-L1 cells were cultured in GM without any inducers at the final differentiation stage.