Human VSMCs (ATCC CRL-1999) were obtained from the American Type Culture Collection. VSMCs were cultured in DMEM (HyClone; GE Healthcare Life Sciences), supplemented with 1% penicillin (100 U/ml)/streptomycin (100 mg/ml) (Beyotime Institute of Biotechnology) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and maintained in a 37°C constant humidified incubator with 5% CO2.
For simvastatin treatment, a total of 5×105 VSMCs/ml were incubated with 2 µM simvastatin (Xingqiong Co., Ltd.) for 24 h following transduction for 24 h with the lentiviral vectors. H19 and p53 expression levels were reduced by transfecting cells with 2 mg shRNAs (lentiviral vectors) targeting H19 (sh-H19) and p53 (sh-p53), respectively, and control cells were transduced with 2 µg negative control vector (sh-NC); all shRNAs were purchased from Shanghai GenePharma Co., Ltd. VSMCs were transduced with sh-p53, sh-H19 or sh-NC in a pGLVH1/GFP+ Puro lentiviral vector (Shanghai GenePharma Co., Ltd.) using Polybrene (4 µg/ml; Merck KGaA).
For simvastatin treatment, a total of 5×105 VSMCs/ml were incubated with 2 µM simvastatin (Xingqiong Co., Ltd.) for 24 h following transduction for 24 h with the lentiviral vectors. H19 and p53 expression levels were reduced by transfecting cells with 2 mg shRNAs (lentiviral vectors) targeting H19 (sh-H19) and p53 (sh-p53), respectively, and control cells were transduced with 2 µg negative control vector (sh-NC); all shRNAs were purchased from Shanghai GenePharma Co., Ltd. VSMCs were transduced with sh-p53, sh-H19 or sh-NC in a pGLVH1/GFP+ Puro lentiviral vector (Shanghai GenePharma Co., Ltd.) using Polybrene (4 µg/ml; Merck KGaA).