5 aminoimidazole 4 carboxamide 1 β d ribofuranoside aicar

The human colorectal cancer cell line Caco-2 (ATCC, Manassas, VA, USA) and the human neuroblastoma cell line SH-SY5Y (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Gaithersburg, MD, USA) at 37 °C in an atmosphere containing 5% CO₂. γ-Aminobutyric acid (GABA) was purchased from Abcam (Cambridge, UK). 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and retinoic acid (RA) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan).

Mouse 3T3-L1 fibroblast cells were used in this study; 3T3-L1 cells were maintained in vitro at 37 °C (5% CO2) in DMEM-HG (Dulbecco’s Modified Eagle’s Medium containing a high concentration (4,500 mg/L) of glucose (D5796; Sigma, St. Louis, MO, USA) supplemented with 75 μg/mL penicillin, 50 μg/mL streptomycin and 10% (v/v) fetal bovine serum (FBS)). DIF-1, DIF-1(3M), and CP-DIF-1 were synthesized as previously described [16 (link)] and stored at –20 °C as 2.5–10 mM solutions in dimethylsulfoxide (DMSO). DNP, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and 6-[4-(2-Piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine (compound C) were obtained from Wako Pure Chemical Industries (Osaka, Japan). DNP were dissolved in ethanol (EtOH) and compound C in DMSO. Rabbit antibodies against AMPKα, phospho-AMPKα, AMPKβ, phospho-AMPKβ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA). Alkaline phosphatase-conjugated goat anti-rabbit IgG antibody, nitroblue tetrazolium (NBT), and 5-bromo-4-chloro-3′-indolylphosphate (BCIP) were purchased from Promega (Madison, WI, USA). The small interfering RNA (siRNA) for AMPKα1/2 (sc-45313) was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).