Isobutylmethylxanthine ibmx

MSCs were grown to full confluency on six-well plates and exposed to adipogenic induction medium (DMEM-LG, 10% FCS, 1% penicillin/streptomycin (Biochrom), 1 µM dexamethasone (Sigma), 60 µM indomethacin (Sigma), 0.5 mM isobutylmethyl-xanthine (IBMX; Sigma), and 5 µg/mL insulin (Sigma)) for 21 days. The medium was replaced every 2-3 days. Oil Red O (Sigma-Aldrich) staining was performed for morphologic examination. Semiquantitative measurements were performed by extracting the ORO dye and measuring the absorbance at a wavelength of 492 nm (Tecan).

To assess the multilineage differentiation capacity, the obtained bone marrow stem cells (BMSC) underwent osteogenic, adipogenic, and chondrogenic induction by different culture media.
For osteogenic differentiation, cells were cultured with osteogenic medium with α-MEM supplemented with 10% FBS, 10⁻⁷ M dexamethasone (Sigma-Aldrich), 10 mM β-glycerol phosphate (Sigma-Aldrich), and 50 mM ascorbate-2-phosphate (Sigma-Aldrich). After 3 weeks of differentiation, the mineralization was stained by Alizarin Red S staining. For adipogenic differentiation, cells were cultured with α-MEM supplemented with 10% FBS, 10⁻⁶ M dexamethasone, 0.5 μM isobutylmethylxanthine (IBMX, Sigma-Aldrich), and 10 ng/mL of insulin (Sigma-Aldrich) for 2 weeks. Lipid accumulation was identified by Oil Red O staining. For chondrogenic differentiation, cells (1 × 10⁶) were seeded in polypropylene tubes with DMEM supplemented with 10⁻⁷ M dexamethasone, 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich), 50 μM ascorbate-2-phosphate, 1 mM sodium pyruvate (Sigma-Aldrich), 50 μg/mL of proline (Sigma-Aldrich), and 20 ng/mL of TGF-β3 (R&D Systems, Minneapolis, MN, USA). After 3 weeks in culture, the pellets were fixed in 10% buffered formalin for 48 h and embedded in paraffin. Then, 4 μm thick sections were processed for toluidine blue staining (Sigma-Aldrich).

The antibodies for KLF16 and PPARγ was purchased from Abcam (Cambridge, MA). Insulin, T3, indomethacin, isobutylmethylxanthine (IBMX) and dexamethasone were purchased from Sigma Aldrich (St. Louis, MO). Dulbecco’ Modified Eagles’ Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Life Technologies (Grand Island, NY)

Samples came from 2-4-week-old C57Bl/6J mice (range of weights = 10-15 g), 5-6-week-old C57Bl/6J mice (15-20 g), and 5-6-week-old C57Bl/6J-ob/ob mice (30-35 g) and were sourced from SiBeiFu Laboratory Animal Technology Company, Beijing, China. Samples from dissections were placed into Dulbecco's modified Eagle medium (Hyclone), with additions of fetal bovine serum (Gibco), penicillin-streptomycin antibiotics, collagenase type I (Sigma), insulin (Sigma), rosiglitazone (Sigma-Aldrich), dexamethasone (Sigma-Aldrich), and isobutyl-methylxanthine (IBMX; Sigma-Aldrich). We used 24-well culture plates (Corning), 96-well culture plates (Corning), a high-fat diet (HFD), Oil-Red-O solution (abcam), paraformaldehyde (Sigma-Aldrich), isopropanol (Sigma-Aldrich), hematoxylin (Sigma), glycogelatin (Solarbio), anti-PPAR-γ (CST), anti-FABP4 (CST), anti-LPL (abcam), anti-UCP1 (CST), F4/80 antibody (BioLegend), CD206 antibody (BioLegend), SDS-PAGE assay products (beyotime), TNF-α, IL-6, IL-1β, IL-10 ELISA kits (MultiSciences), Cholesterol Assay Kit-HDL, LDL/VLDL (abcam), anti-LPA (abcam), anti-ITGAM (CST), and anti-NF-κB (CST).

hMSCs were seeded into four-well plates and exposed to adipogenic induction media composed of DMEM, 10% fetal bovine serum (FBS), 10% horse serum (Gibco, U.S.A.), 100 µM dexamethasone (Sigma, U.K.), 1 µM Rosiglitazone (BRL) (Novo Nordisk Bagsvaerd, Denmark), 3 µg/ml insulin (Sigma, U.K.), 450 µM isobutylmethylxanthine (IBMX) (Sigma, U.K.), and 1% penicillin-streptomycin (Sigma, U.K.) supplemented with FAK inhibitors (PF-573228 and PF-562271) or IGF-1R/InsR inhibitors (NVP-AEW541 and GSK1904529A), which were purchased from Selleckchem Inc. (Selleckchem Inc., Houston, TX, U.S.A.). Inhibitors were used at 5 µM throughout all experiments. Adipocyte induction medium (AIM) was changed every 2 days and for 7 days. Previous published work from our group indicated day 7 as a time point on which adipogenic markers were up-regulated significantly in an enriched population of 70–80% of adipogenic populations [8 (link)].

Mouse mesenchymal cells (D1, ATCC CRL-12424) were grown and maintained routinely in standard medium containing low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine and 1% non-essential amino acids. When cells reached 80% confluence, adipogenic differentiation was induced with adipogenic (‘AD’) medium prepared with standard medium supplemented with 1 μM dexamethasone (Cayman Chemicals, USA), 100 µM isobutylmethylxanthine (IBMX) (Sigma-Aldrich, UK), 1 μM rosiglitazone (Cayman Chemicals, USA), and 10 μg/ml insulin (Sigma-Aldrich, UK). All supplements were prepared according to the manufacturer’s guidelines. AD medium was used throughout the treatment period, as previously described^{55 (link),56 (link)}. Experimental cultures were treated for 9 days in two groups: cells differentiated under standard cell culture thermal conditions (37 °C), and cells differentiated at 32 °C^{57 (link)–59 (link)}. Matching undifferentiated control cells maintained in standard medium were also grown at 37 and 32 °C. The same protocol was applied to human adipose-derived stem cells (hADSCs) (ThermoFisher UK, Cat. No R7788-115) and primary mouse BM-derived stem cells. For the analyses performed in this study, 3 batches of cells were analysed.