Tug 891

ATDC5 chondrogenic cell line was purchased from Sciencell and cultured in a differentiation medium containing DMEM/F-12 (1:1) with GlutaMAX I (Gibco, USA) supplemented with 5% FBS (Gibco, USA), 1% sodium pyruvate, and 0.5% gentamicin (Gibco, USA) for 1 week prior to experimentation. MIN6 cells were stored in a humidified incubator containing 5% CO₂ at 37 °C. The culture media was replaced with fresh media every second or third day. To test the expression of GPR120, the cells were exposed to 10 ng/ml IL-1β for 12, 24, and 48 h. For subsequent experiments, the cells were exposed to 10 ng/ml IL-1β (R&D systems, USA) in the presence or absence of 50 μM GW9508 (Cayman Chemical, USA) or 10 μM TUG891 (Cayman Chemical, USA). H89 (10 μM) (Sigma-Aldrich, USA) was used in the CREB inhibition experiment, and AH7614 (1 μM) (Cayman Chemical, USA) was used in the GPR120 inhibition experiment.

The Raw264.7 cell line (ATCC, Manassas, VA), a mouse monocyte/macrophage cell line, was used due to endogenous expression of functional GPR120 [11 (link)]. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum. In some experiments using EPA, DMEM without phenol-red (FUJIFILM Wako Pure Chemical Corporation) was used. Cells were pre-treated with GPR120 agonists, EPA (Mochida Pharmaceutical Co., Ltd.) or TUG-891 (Cayman Chemical, Ann Arbor, MI), before stimulation with lipopolysaccharide (LPS) (Lot. 123M4052V, # L2654, Sigma Aldrich).