Ambion silencer select validated sirna

Human aortic SMCs and adventitial fibroblasts were purchased from Promo Cell (Catalog No: C-12533, C-12380) and cultured in growth medium (Promo Cell, Catalog No: C39262). The SMC and fibroblasts cell purity were verified using a SMC α-actin (1:100, Abcam catalog No: ab5694) and ERTR7 (1:100, Abcam catalog No: ab5694) antibodies by immunohistochemistry. Cells of passages 2–3 were used for the short interfering (si)RNA transfection study. SMCs were transfected with control siRNA or siRNA targeting human calpain-2 (Ambion-Silencer Select validated siRNA, ThermoFisher Scientific) sequences using RNAiMax lipofectamine transfection reagent (Life Technologies, ThermoFisher Scientific). After 48 h of transfection, cells were incubated with either vehicle or AngII (1 μM) for 24 h; then protein lysates were harvested for western blot analyses.

Rat aortic SMCs were isolated and cultured as described previously.^{20 (link)} Briefly, rat aortas were excised and dissected free of adventitia and fat. The vessels were further digested by collagenase type I (Worthington Biochemical, 1 mg/ml) and elastase type III (sigma 0.125 mg/ml) at 37°C in a CO₂ humidified chamber. Then, cell suspensions were centrifuged and the cell pellets were re suspended and grown in DMEM with 10% fetal bovine serum and penicillin and streptomycin (1%) in 5% CO₂ at 37°C. The SMC phenotype was determined as described above. Cells of passages 2–3 were used for the short interfering (si)RNA transfection study. SMCs were transfected with control siRNA or siRNA targeting rat calpain-2 or filamin A (Ambion - Silencer Select validated siRNA, ThermoFisher Scientific) sequences using RNAiMax lipofectamine transfection reagent (ThermoFisher Scientific). After 48 h of transfection, cells were treated with either vehicle or AngII (100 nM) for 24 h and then harvested for western blot analyses.