AngII-induced Ch25h expression in VSMCs

Before the experiments, the VSMCs were serum deprived overnight using DMEM supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich). VSMCs were pretreated with either dimethyl sulfoxide (DMSO) vehicle or various inhibitors separately: 10 µM candesartan, 1 µM JNK-IN-8 (IN-8), 50 µM SB202190, 20 µM PD98059 (Sigma-Aldrich), 1 µM YM-254890 (Wako Chemicals), and 5 µM DPI (Sigma-Aldrich). Pretreatment lasted for 30 min. Following pretreatment, VSMCs were stimulated with 100 nM AngII or vehicle for 1 h. In the other sets of experiments, the VSMCs were stimulated with either vehicle or 3 µM TRV120023 (TRV3; Proteogenix) for 1 h. To assess time dependency of Ch25h mRNA expression, VSMCs were stimulated with 100 nM AngII for 1, 2, 3, 4, 5, and 6 h or not stimulated.
For the detection of 25-HC, VSMCs were stimulated with 1 µM AngII for 2, 4, 8, 16, and 24 h or not stimulated. The experiments were performed in duplicates. After the hormone stimulation, the supernatants were collected and subjected to LC-MS/MS measurement.