Rasmcs

Rat aortic smooth muscle cells (RASMCs) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in the conditions described in our previous report [9 (link)]. The characteristics and contractile phenotype of VSMCs are maintained up to passage 15, which were confirmed by immunofluorescence staining for alpha-smooth muscle actin (α-SMA) (Figure S1). For experiments, the cells were used at passages 5–10.

RASMCs were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in SMC medium (ScienCell, Cat#: 1101), containing 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin. RASMCs within passage 5 to 12 were used for all experiments. RASMCs were stimulated with 100 nM Ang II (Sigma, Cat#: A9525-50MG) for 24 h before harvest. To knockdown PDE4D, PDE4D siRNA (Ribobio, siB180730051733) and control siRNA (Ribobio, siN0000001-1-5) were purchased from Ribobio (Guangzhou RiboBio Co., Ltd., Guangzhou, China). RASMCs were transfected with 200 nM PDE4D siRNA in 5 μl of Oligofectamine (Invitrogen, Carlsbad, CA, USA, Cat#: 12252011) for 48 h. The siRNA transfection efficiency was determined by real-time polymerase chain reaction (RT-PCR), western blot and immunofluorescence assay (Supplementary Fig. 5a–e).