2.5 | Aortic SMC culture
Aortic SMCs were isolated from 8-week-old male WT and SKI mice as previously described (Tong et al., 2015) and cultured in DMEM supplemented with 10% FBS (ExCell Bio, Cat# FSP500), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2. SMC phenotype was confirmed by α-smooth muscle actin immunostaining. Cells from passages 3 to 8 were used. All SMCs used were isolated from C57BL/6J background due to the SMCs from LDLR−/− background were infeasible for further studies after subculture. Using different genetic background for mechanism study is commonly used in literature (Nakao et al., 2017). In some experiments, SMCs were administrated with pioglitazone (10 μM, MCE, Cat# U72107), or pyrrolidinedithiocarbamic acid (PDTC, ammonium salt, 10 μM, Macklin, Cat# A800469) for 48 hr, before being collected for Western blot analysis or cell function studies. DMSO served as a solvent control, and its final concentration was less than 0.1%.