S8244

ECAR and OCR measurement were performed using a Seahorse XFe24 analyzer (Seahorse Bioscience) on MEFs (20,000 cells/well) plated on XFe24 tissue culture plates coated with fibronectin (3 μg/ml). Seahorse XF Cell Mito Stress Test Kit (103015-100), XF Glycolytic Rate Assay Kit (103344-100), XF Real-Time ATP Rate Assay Kit (103592-100), and XF Palmitate Oxidation Stress Test Kit (103693-100) were used according to the manufacturer’s protocol. For OXPHOS experiments testing glucose, glutamine, and fatty acid as respiratory substrate [44 (link)], cells were incubated with DMEM minimum medium (without glucose, sodium pyruvate, L-Glutamine, and 1% FBS) 24 h before the assay. One hour prior to measurements, cells were treated/untreated, respectively, with UK5099 (10 µM, Selleckchem, S5317), BPTES (20 µM, Selleckchem, S7753), or Etomoxir (100 µM, Selleckchem, S8244), and incubated (CO₂-free atmosphere) at 37 °C.

Primary LF were isolated from 6-week-old mice as previously described [20 (link)] and cultured with DMEM containing 15% FBS at 37 °C. Passage 2 cells were treated with MitoQ (50 nM, HY-100116, MCE), fenofibrate (10 μM, T1149, Topscience), oleic acid (10 μM, S4707, Selleck), etomoxir (50 μM, S8244, Selleck), bafilomycin (5 nM, S1413, Selleck) and SRT1720 (4 μM, S1129, Selleck) (Additional file 1). The dosages of these compounds were based on published papers. And MTT test for them was performed (Additional file 2).