Nefa detection kit

The content of NEFA was measured using a NEFA Detection kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan), following the manufacturer's protocol. Briefly, cells were treated with drugs and grouped as aforementioned. Following treatments, 22 µM [9,10-³H]oleate was added to cells for 4 h. Cells were subsequently washed two times with PBS and cultured in DMEM for 4 h. The cells were digested with 0.25% EDTA-trypsin (Beijing Solarbio Science & Technology Co., Ltd.), resuspended in DMEM and centrifuged with 1,000 × g for 10 min. The supernatant was transferred into a clean centrifuge tube. Lipid was extracted from supernatant using N-hexane: Isopropanol solution (3:2) and a thin layer chromatography silica gel plate (25 (link)). The silica gel of containing NEFA fraction was scraped from the silica gel plate and dissolved in N-hexane: Isopropanol solution (3:2). Scintillation solution was added and the ³H-labeled NEFA protein content in the cells was measured using a L6500 liquid scintillation counter (Beckman Coulter, Inc., Brea, CA, USA). NEFA protein concentration was detected using a Bradford protein assay kit (Beyotime Institute of Biotechnology, Haimen, China).