The human laryngeal cancer cell line Hep-2 was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 μg/ml penicillin/streptomycin at 37°C with 5% CO2.
ACE gene were cloned from human genomic DNA into the pGL3 basic vector (Promega, E1751). The cells were seeded in 96-well plates. When the concentration reached 80~90%, the cells were transfected with plasmids pGL3 containing ACE and empty plasmids pGL3 using Lipofectamine 2000 (Invitrogen 11668027) according to the manufacturer’s instructions. Moreover, in order to reinforce the role of ACE on the progression of laryngeal cancer, some of the Hep-2 cells transfected with empty plasmids pGL3 were treated with Fosinopril (Sigma) at the concentration of 10 μmol/L for 48 h. The experiment was performed in triplicate.
ACE gene were cloned from human genomic DNA into the pGL3 basic vector (Promega, E1751). The cells were seeded in 96-well plates. When the concentration reached 80~90%, the cells were transfected with plasmids pGL3 containing ACE and empty plasmids pGL3 using Lipofectamine 2000 (Invitrogen 11668027) according to the manufacturer’s instructions. Moreover, in order to reinforce the role of ACE on the progression of laryngeal cancer, some of the Hep-2 cells transfected with empty plasmids pGL3 were treated with Fosinopril (Sigma) at the concentration of 10 μmol/L for 48 h. The experiment was performed in triplicate.