3 isobutyl 1 methylxanthine

For lipid deposition, primary hepatocytes were incubated with a 1.2 mM fatty acid solution [oleic acid (OA, Sigma-Aldrich): palmitic acid (PA, Sigma-Aldrich) = 2:1] in William’s Medium E (Gibco) containing 15% FBS (Gibco) for 24 h (n = 3, three technical repetitions were performed for each sample). The primary preadipocytes were incubated with a cocktail of insulin (10 μg/mL, Sigma-Aldrich), dexamethasone (1 μM, Sigma-Aldrich), and 3-isobutyl-1-methylxanthine (0.5 mM, Sigma-Aldrich) in DMEM/F12 (Gibco) with 10% FBS for 2 d, followed by culture with DMEM/F12, 10% FBS, and insulin (10 μg/mL, Gibco) for another 2 d, and then cultured with DMEM/F12 and 10% FBS for 2 d (n = 3, three technical repetitions were performed for each sample) [26 (link)]. The medium was replaced with DMEM/F12 supplemented with 10% FBS for 2 d.

3-(4,5-Dimethylthiazol-2-y1)-2,5-dipheny-ltetrazolium bromide (MTT), 3-isobutyl-1-methylxanthine (IBMX), and oleic acid were purchased from Sigma-Aldrich (Cat. Nos. M2128, I7018, and O1383, St. Louis, MO, USA). Dexamethasone (Dex) was obtained from Sango (Cat. No. A601187, Shanghai, China), and insulin was obtained from Shanghai Yuanye BioTechnology Co., Ltd. (Cat. No. S31559, Shanghai, China). The cell culture medium, trypsin, penicillin–streptomycin, and fetal bovine serum (FBS) were from Gibco (Burlington, MA, USA). Oil Red O was obtained from Solarbio (Cat. No. G1260, Beijing, China). Malondialdehyde (MDA) assay kits were obtained from Dojindo (Cat. No. M496, Kumamoto, Japan). The glutathione assay kit and reactive oxygen species (ROS) assay kit were bought from Beyotime (Cat. Nos. S0053 and S0033, Nanjing, China). The insulin ELISA kit was purchased from Millipore (Cat. Nos. RAB0817, Millipore, Bedford, MA, USA). Primary antibodies against SLC7A11/xCT (26864-1-AP), TRF (17435-1-AP), and GPX4 (14432-1-AP) were purchased from Proteintech (Rosemont, IL, USA). Anti-AMPKα (D63G4) (5832), anti-phospho-AMPKα (Thr172) (40H9) (2535), anti-ACC (C83B10) (3676), and anti-phospho-ACC (Ser79) (D7D11) (11818) were obtained from Cell Signaling Technology (Danvers, MA, USA). ACSL4 was from Abcam (ab155282, Cambridge, MA, USA) and β-actin was from HUABIO (EM21002, Shanghai, China). BODIPYTM 581/591 C11 was obtained from Thermo Fisher Scientific (Cat. No. D3861, Waltham, MA, USA). Ferrostatin-1 and Erastin were purchased from MedChemExpress Technology (Cat. Nos. HY-100579 and HY-15763, Monmouth Junction, NJ, USA).

Trilineage differentiation (AT-MSC, n = 6; BM-MSC, n = 4) was performed as previously described except where indicated [18 ]. Briefly, for adipogenesis and osteogenesis, cells were cultured for 14 days with either EM as described above or induction medium. Adipogenesis induction medium consisted of low-glucose DMEM with 1 μM dexamethasone (Sigma-Aldrich, St. Louis,Missouri), 0.5 mM 3-isobutyl-1-methyl-xanthine (Sigma-Aldrich, St. Louis, Missouri), 10 μg/mL recombinant human (rh) insulin (Sigma-Aldrich, St. Louis, Missouri), 0.2 mM indomethacin (Sigma-Aldrich, St. Louis, Missouri), 15% rabbit serum (Sigma-Aldrich, St. Louis, Missouri), 1% L-glutamine, and 1% antibiotic antimycotic solution (ABAM, Sigma-Aldrich, St. Louis, Missouri). Osteogenesis induction medium consisted of low-glucose DMEM with 0.1 μM dexamethasone, 10 mM glycerol 2-phosphate, 0.05 mM ascorbic acid, 10% FBS, 1% L-glutamine, and 1% ABAM. For chondrogenesis, 250,000 cells were pelleted in a 96-well plate and cultured for 21 days in high-glucose DMEM (Lonza, Walkersville, Maryland), 0.1 μM dexamethasone, 0.1 mg/mL ascorbic acid (Sigma-Aldrich, St. Louis, Missouri), 10 ng/mL TGF-β3 (R&D Systems, Minneapolis, Minnesota), 200 mM Glutamax (Life Technologies, Grand Island, New York), 10 mg proline (Sigma-Aldrich, St. Louis, Missouri), 40 μg/mL ascorbic acid, 100 mM sodium pyruvate (Life Technologies, Grand Island, New York), 1% Insulin-Transferrin-Selenium (Life Technologies, Grand Island, New York), 1% L-glutamine, and 1% ABAM. To promote better chondrogenesis, 0, 50, 100, or 200 ng/mL bone morphogenic protein 2 (BMP-2) was added to the media.
Adipogenesis and osteogenesis samples were stained with Oil Red O and Alizarin Red S stains (Sigma-Aldrich, St. Louis, Missouri) respectively. Chondrogenesis samples were histologically evaluated with toluidine blue staining for glycosaminoglycan content and hematoxylin and eosin staining for general pellet structure as previously reported [36 (link)]. Adipogenic, osteogenic, and chondrogenic mRNA transcript abundance was analyzed by RT-qPCR using the primers listed in Table 2. cDNA was synthesized from 500 ng RNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, New York) using manufacturers' instructions. PCR reactions were performed using the PerfeCta SYBR Green FastMix, ROX (Quanta BioScience, Gaithersburg, Maryland) with the Applied Biosystems 7300 Real Time PCR system. Data were analyzed using the 2^-ΔΔCT method. Gene expression data is presented as the induction medium-treated cultures relative to the expansion medium-treated control cultures with GAPDH used as reference gene.

Primary bone marrow-derived cells were induced to osteogenesis in the standard osteogenic medium composed by α-MEM 10% FCS supplemented with 50 μg/ml ascorbic acid (Sigma-Aldrich Corporation, St Quentin Fallavier, France) and 10 mM β-glycerophosphate (Sigma-Aldrich corporation) for up to 14 days and the medium was changed every 2 or 3 days.
Adipogenesis was induced in the standard adipogenic medium containing Dulbecco’s Modified Eagle Medium (DMEM) (PAN Biotech) 10% FCS supplemented with 10 μg/ml insulin/0,1 or 0,5 μM dexamethasone (depending on the experiment)/100 μM indomethacin/500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corporation) for 4 days and then maintained in 10 μg/ml insulin/0,1 or 0,5 μM dexamethasone/5 μM pioglitazone (Sigma-Aldrich Corporation) for 10 days and the medium was changed every 2 or 3 days.
In order to obtain both adipocytes and osteoblasts in the same differentiation medium, cells were cultured in the standard osteogenic medium supplemented with different concentrations of Dex ranging from 50 nM to 150 nM. For these experiments, co-differentiation medium referred to osteogenic medium added with 100 nM of Dex.