3 isobutyl 1 methylxanthine

Spermine tetrahydrochloride, silver nitrate (AgNO₃), sodium borohydride (NaBH₄), adenosine 5′-triphosphate disodium salt hydrate (ATP), protein kinase A (PKA, from bovine heart), and dithiothreitol (DTT) were purchased from Sigma–Aldrich (Shanghai, China). Dihydrochloride hydrate (H-89) was obtained from J&K Scientific Ltd. (Shanghai, China). Protein kinase B (PKB) was purchased from Bio-Techne China Co., Ltd. (Shanghai, China). Forskolin, 3-isobutyl-1-methylxanthine (IBMX), and Tris-HCl buffer solution (pH 7.5) were purchased from Sangon Biotech (Shanghai, China). Substrate peptide for PKA (TAMRA-LRRASLG, 98%), and phosphorylated substrate peptide were purchased from GL Biochem (Shanghai, China). Magnesium chloride (MgCl₂), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), glycerol, and dimethyl sulfoxide (DMSO) were obtained from Shanghai Chemicals Ltd. (Shanghai, China). The human cervical carcinoma cells (HeLa) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All other reagents were of A.R. grade and used as received without further purification. Ultrapure water with a resistivity of 18.2 MΩ·cm was used.

3T3-L1 (ATCC CL-173) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) in a 37 °C incubator containing a 5% CO₂ atmosphere. For adipocyte differentiation, 3T3-L1 preadipocytes were trypsinized and plated in a 12-well plate. Cells were cultured for an additional day after reaching confluency, and 10 mg/mL insulin (Sangon Biotech, Shanghai, China), 1 µM dexamethasone (Sigma, St. Louis, MO, USA), and 0.5 mM 3-isobutyl-1-methylxanthine (Sangon Biotech, Shanghai, China) were added to the culture medium for 2 days. The medium was then replaced with the basic culture medium containing only 10 mg/mL insulin for another 2 days. From Days 4 to 7, the cells were maintained in basic culture medium and were additionally administrated 100 ng/mL DON and 1 µM rosiglitazone (RGZ) (MedChemExpress, Princeton, NJ, USA) from Days 0 to 7 during differentiation. The DON and RGZ were replenished every two days.