3 isobutyl 1 methylxanthine ibmx

With osteogenic differentiation medium, bone marrow-derived mesenchymal stem cells (BMSCs) were planted onto 24-well plates in the presence 0, 10, 20, and 40 μM MUS. Cells were washed 3 times with PBS, and were fixed with 4% paraformaldehyde for 15 min. It was 14 days and 21 days after the osteogenic induction that ALP staining (Sigma‐Aldrich) and ARS staining (Nanjing Jiancheng Chemical Industrial Co., Nanjing, China) were conducted, respectively.
Furthermore, to induce adipogenesis, BMSCs were cultured with 10% FBS α‐MEM supplied with 10 μg/mL insulin, 200 μmol/L indomethacin, 1 μmol/L dexamethasone, and 0.5 mmol/L 3‐isobutyl‐1‐methylxanthine (IBMX) (Cyagen Biosciences). Differentiated cells were then marked with Oil Red O staining (Sigma‐Aldrich).
Staining cells were observed under a microscope (Leica, Germany), and the mineralized area was analyzed by ImageJ software (National Institutes of Health, USA).

To identify the role of l‐THP on osteogenesis and the formation of the calcified nodule, we flushed bilateral femoral bone marrow of 4‐week‐old C57BL/6 mice to isolate bone marrow mesenchymal stem cells (BMSCs). To induce osteogenesis, BMSCs were cultured with complete medium supplied with 100 nmol/L dexamethasone, 50 μmol/L ascorbic acid and 10 mmol/L β‐glycerophosphate (Cyagen Biosciences). Prepared cells were stained with ALP staining (Sigma‐Aldrich) after osteogenic induction for 14 days, while alizarin red staining was conducted after 21 days. To induce adipogenesis, BMSCs were cultured with 10% FBS α‐MEM supplied with 10 μg/mL insulin, 200 μmol/L indomethacin, 1 μmol/L dexamethasone and 0.5 mmol/L 3‐isobutyl‐1‐methylxanthine (IBMX) (Cyagen Biosciences). Differentiated cells were then marked with Oil Red O staining (Sigma‐Aldrich).

hBMSCs (Cyagen Biosciences, Guanzhou, China) and human liposarcoma SW872 cell line (ATCC, Rockville, MD, USA) were cultured in modified Eagle’s medium (MEM) containing 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM l-glutamine at 37 °C under a humidified, 5% CO₂ atmosphere. Culture medium was changed every 2–3 days. When the cells were 100% confluent, they were induced to differentiate into adipocytes with adipogenic differentiation medium containing Human Mesenchymal Stem Cell Adipogenic Differentiation Basal Medium A, Mesenchymal Stem Cell-Qualified Fetal Bovine Serum (10%), glutamine (2 mM), penicillin (100 units /mL), streptomycin (0.1 mg/mL), insulin (10μg/mL), 3-isobutyl-1-methylxanthine (IBMX) (500 μM), rosiglitazone (0.5 μM), and dexamethasone (1 μM) (Cyagen Biosciences, Guanzhou, China). ATRA, 9CRA, 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propen-1-yl] benzoic acid (TTNPB), and SR11237 (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) for the experiments. The cells were cultured under dim light when treated with retinoids to prevent their degradation.