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%.