Proteomics of HTR8 cells with bile acids and EBSS treatment

HTR8 cells maintained in Earle's balanced salt solution (EBSS) and treated with 100 μM chenodeoxycholic acid (CDCA) or 100 μM deoxycholic acid (DCA) for 18 h were subjected to proteomic analysis After treatment with bile acids (CDCA or DCA) and EBSS, HTR8 cells (two duplicates in each group) were harvested and lysed. The protein lysates of HTR8 cells were digested with trypsin (Promega, Madison, WA, USA). The resulting peptide in each sample was then labeled using a Tandem Mass Tags (TMT) Labeling kit (ThermoFisher Scientific) following the manufacturer’s instructions and fractionated by reverse-phase chromatography using high-performance liquid chromatography (1260 Infinity II; Agilent, USA). Liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was performed on a mass spectrometer (positive ion mode; Q Exactive Plus; ThermoFisher Scientific) coupled with a nanoflow LC system (Easy-nLC; ThermoFisher Scientific). MS data were acquired and processed using MASCOT (version 2.6; Matrix Science, London, UK) embedded into Proteome Discoverer 2.2. Proteins with fold changes higher than 1.2 or less than 0.83 and p values less than 0.05 were considered as differentially expressed.