Aicar

A stock solution (100 mM) of palmitic acid (#P0500, Sigma-Aldrich, Munich, Germany) was prepared in DMSO and was added at a final concentration of 6 mM to FCS (containing 5% serum albumin) or modified Krebs-Ringer buffer (KRB) supplemented with 5% BSA. The GPR40 agonist TUG-469 (a kind gift from Prof. T. Ulven, Southern University of Denmark, Denmark) was applied at concentrations of 3–10 μM in culture medium supplemented with 1% FCS (containing 0.05% BSA) [2 (link), 18 (link)]. The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO. Inhibitors were applied 1 h prior to palmitate and kept throughout the treatment.

C2C12 skeletal myoblasts were cultured in growth medium (GM; DMEM supplemented with 20% FBS). Before confluency the GM was replaced with differentiation medium (DM; supplemented with 2% horse serum) with or without AICAR (Enzo Life Sciences). As for SCs isolation, the manufacturer (Miltenyi Biotec)’s instructions were followed (Gatta et al., 2017 (link)). The purified cells were then resuspended in DMEM supplemented with 10% FBS, 20% HS, and 3% CEE, and seeded in collagen precoated dishes.

DMSO (Sigma Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS, Corning, NY, USA), Compound C (ComC, an AMPK inhibitor, Enzo Life Sciences, Farmingdale, NY, USA), AICAR (AMPK activator, Enzo Life Sciences, Farmingdale, NY, USA), and Sn(IV) protoporphyrin IX dichloride (SnPP, HO inhibitor, Frontier Scientific, Logan, UT, USA) were bought. KRGE containing various ginsenosides was obtained from the Korea Ginseng Cooperation (Daejeon, Korea) and stored at 4 °C. Next, a 0.2 g/mL stock solution prepared in filtered distilled water was aliquoted and stored with light protection at −25 °C.