Tissue culture media, serum, phosphate-buffered saline (PBS), and additives were purchased from Hyclone (South Logan, UT, USA). Murine pre-adipocytes (3T3-L1, American Type Culture Collection; Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% newborn calf serum and antibiotics. For adipocyte differentiation induction, pre-adipocytes were plated at 24-well plates. After confluence, the cells were incubated in DMEM plus 10% fetal bovine serum, 1 mM dexamethasone, 0.5 mM 3-iso-butyl-1-methylxantine (IBMX), and 1 mg/ml insulin for 2 days followed by treatment with insulin alone. The medium was replaced every 2 days for 8–14 days. 3T3-L1 cells (5000 cells/well) were incubated with 5 or 10 nM namodenoson (Can-Fite BioPharma Ltd, Petah Tikva, Israel) or vehicle at a 96-well plate for 48 h, and cell proliferation was assessed by the 3H-thymidine incorporation assay (Sanquine, Amsterdam, The Netherlands).

For lipid accumulation analysis, pre-adipocytes were incubated in the differentiated medium (high-glucose DMEM high with 10% fetal bovine serum and 10 mg/ml insulin, 1 mM dexamethasone and 0.5 mM IBMX), and the cells were treated with 5 or 10 nM namodenoson or vehicle for 48 h. Accumulation of lipid droplets was evaluated by Oil-Red-O staining (Sigma Aldrich, St. Louis, Missouri, USA). The cells were washed with PBS and incubated with 3.7% formaldehyde in H2O (Sigma Aldrich, St. Louis, Missouri, USA) for 1 h, followed by incubation with Oil-Red-O solution for 45 min. Cells were washed, visualized, and photographed under an Olympus microscope (Tokyo, Japan). Cells were dissolved in isopropanol to quantify lipid accumulation, and the optical density was read on a Dynatech Corp. Microelisa reader (Chantilly, VA, USA) at 595 nm. Experiments were performed in triplicates; data are presented as mean ± SD. Statistical significance was determined by t-test. P < 0.05 was considered statistically significant.

Western blot analyses were performed with culture conditions as in the 3H-thymidine experiments with 5 or 10 nM namodenoson or vehicle. After incubation for 24 h, the cells were rinsed with ice-cold PBS and transferred to ice-cold radio-immunoprecipitation assay buffer with 1× protease & phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA) for 20 min. Cell debris was removed by centrifugation at 4 °C for 10 min, at 7500 × g. The supernatant was used for Western blot analyses. Protein concentrations were determined using the NanoDrop assay (ThermoFisher Scientific, Waltham, MA, USA). Fifty micrograms were separated by 12% SDS-PAGE (ThermoFisher Scientific, Waltham, MA, USA), and electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with 1% bovine serum albumin and incubated with the relevant primary antibodies (dilution 1:1000, all purchased from Santa Cruz Biotechnology, Dallas, TX, USA, catalog numbers are detailed in Supplementary Table 1) for 24 h at 4 °C. Blots were washed and incubated with the secondary antibody for 1 h at room temperature. Bands were recorded using a BCIP/NBT kit (Promega Madison, WI, USA).

All laboratory animal procedures were evaluated and approved by The Hebrew University Institutional Animal Care and Use Committee (approval #MD-21-16593-3) and followed the guidelines for laboratory animal welfare. Mice studies utilized C57BL/6 J males, 4–6 weeks old (The Jackson Laboratory, stock 000664, Bar Harbor, ME, USA). The mice (6 in each treatment group) were fed a high-fat diet (D12492, 60 kcal% fat; Research Diets, Inc., New Brunswick, NJ, USA) or a regular diet (“lean diet group”). After 12 weeks on this diet, mice received daily namodenoson (100 μg/kg, oral) or remained untreated. Weight was measured weekly from treatment initiation up to week 16. Data are presented as mean±SD. The difference in weight between the groups was determined by t-test. P < 0.05 was considered statistically significant.