Roswell park memorial institute (rpmi)

Canagliflozin was provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Streptozotocin and AICAR were purchased from Wako (Osaka, japan). Nicotinamide was purchased from SIGMA (St. Lewis, MO, USA). 2-Deoxyglucose (2-DG) was purchased from Tokyo Chemical Industries (Tokyo, Japan). Compound C was purchased from Calbiochem (CAS 866405-64-3). The cell culture reagents RPMI and DMEM were purchased from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Biosera (Kansas City, MO, USA).
Antibodies were obtained from Cell Signaling Technology (pACC-11818S, ACC-3662S, pAMPK-4188S, AMPK-5832S, Cyclin D1-2922S, p-p70-9234S, p-70-2708S), R&D (PDGFRβ) and Santa Cruz (actin sc-47778 F1417, Pin-1sc46660 B0917).

Wild–type, TLR2-/-, TLR4-/- and MyD88-/- mice (on BALB/c background) were purchased from Oriental Bioservice, Inc (Kyoto, Japan). Femurs were removed at 8 weeks of age, the soft tissue removed, and flushed with Hanks to recover the bone marrow. BMDMs were cultured on plastic dishes in RPMI (Nissui, Tokyo, Japan) containing penicillin/streptomycin (Gibco, New York, NY) and 10% inactivated fetal bovine serum (Thermo Scientific, South Logan, UT). 10 ng/ml of GM-CSF (PeproTech EC, London, UK) was added to the culture on days 0 and 3. After 6 days in culture, the adherent BMDMs were collected by adding 0.02% 5 ml EDTA and then scraping the loose cells off. Then BMDMs were re-plated at a concentration of 3 × 10⁵ cells/ml into 24-well plates at 1 ml/well. BMDMs were incubated with PBS or LPS (final 1 μg/ml) for 12 h. Cytokines secreted into the culture medium by BMDMs were measured by ELISA.

Human THP-1 monocytes and RAW 264.7 murine macrophages cells were purchased from Riken Cell Bank. The THP-1 cells were cultured in RPMI supplemented with 10% FBS (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 37 °C in 5% CO₂. The THP-1 cells were passaged every 2–4 days at a ratio of 1:5–10. In the experiment, THP-1 cells were differentiated into macrophage-like cells by adding 1 μM PMA (Sigma–Aldrich Corp., St. Louis, MO, USA) to the cells at the time of seeding. The RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS (Nissui Pharmaceutical Co., Ltd.) at 37 °C in 5% CO₂. The RAW 264.7 cells were passaged every 2–4 days at a ratio of 1:5–10.