Human asm cells

Human ASM cells were purchased from ScienCell (Corte Del Cedro Carlsbad, CA, USA). E. coli BL21(DE3) and the pET28(a)+ vector were purchased from Novagen (Germany). The recombinant human CTGF/CCN2 C-terminal domain, Cyr61/CCN1 and NOV/CCN3 were purchased from PROSPEC (USA). The monoclonal mouse anti-human CTGF/CCN2 C-terminus, Cyr61/CCN1 and NOV/CCN3 monoclonal antibody were purchased from R&D system (USA). The anti-6xHis monoclonal antibody was from abcam (UK). The Cell_Light EdU DNA Cell Proliferation Kit was from Ribobio (China). LY294002 (PI3K inhibitor), rabbit antibodies against Akt, mTOR, β-actin, phosphorylated-Akt (Ser473), and phosphorylated-mTOR, and HRP-goat anti-rabbit IgG were purchased from Cell Signaling (USA). NcoI, XhoI, T4 DNA ligase and Easy Taq DNA polymerase were purchased from TaKaRa (Japan). The BCA protein assay kit was provided by Tiangen (China). Cell culture media were purchased from Gibco BRL (USA). The newborn calf serum was obtained from Shiqing (China). Isopropyl-1-thio-β-D-galactoside (IPTG) was purchased from Sigma-Aldrich. The His-bind resin column and total protein extraction kit were purchased from KeyGEN (China).

Human ASM cells were obtained from ScienCell Research laboratories. Cells were cultured in six-well plates in Smooth Muscle Cell Medium (SMCM) containing 10% FBS and were maintained at 37 °C and 5% CO₂ as previously described [13 (link)]. Cells from passage 3–6 maintained their SMC phenotype and were used in all experiments. ASM cells were characterized by smooth muscle cell markers including smooth muscle α-actin and smooth muscle heavy chain using immunofluorescence. In inhibition experiments, inhibitors of signal transduction pathways were added for 2 h before the addition of VEGF. All inhibitors were dissolved in dimethyl sulfoxide (DMSO; final concentration of 0.1%, vol/vol) and added to the medium. Vehicle controls contained the same amount of DMSO.