Paecs

PAECs were purchased from Lonza Group, Ltd. In strict accordance with the protocol, the cells were cultured with 10% FBS and high-glucose Dulbecco's modified eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) and observed under a light microscope. The cells at passage 3 were detached with trypsin, inoculated into a 24-well plate at a density of 2×10⁶ cells/well and cultured until they grew into monolayers. When the cell density reached 75%, the cells were considered to be in logarithmic phase and were inoculated into a 6-well cell culture plate. After 12 h, according to the protocol of Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), the cells were transfected with NC mimic, let-7d mimic, NC inhibitor or let-7d inhibitor plasmids (final concentration, 50 nM) for 48 h (22 (link)). The cells were then assigned to the following groups: The blank group (without transfection of any plasmid), the NC mimic group (transfected with NC mimic plasmid), the let-7d mimic group (transfected with let-7d mimic plasmid), the NC-inhibitor group (transfected with NC inhibitor plasmid) and the let-7d inhibitor group (transfected with let-7d inhibitor plasmid). The transfection plasmid was constructed by Invitrogen; Thermo Fisher Scientific, Inc.

PAECs (Lonza, Basel, Switzerland) were cultured at 37 °C in a 5% CO₂ incubator in endothelial cell growth medium-2 (Lonza), 1% penicillin–streptomycin (Welgene, Daegu, Republic of Korea), and MycoZap (Lonza). For the experimental treatments, PAECs were grown to 70–90% confluency. Lenti-HEK293X cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin–streptomycin (Welgene). Small interfering RNA (siRNA) (Stealth siRNA; Invitrogen, Grand Island, NY, USA) was transfected using Lipofectamine RNAi MAX (Invitrogen) or DharmaFECT4 according to the manufacturer’s instructions. Plasmid DNAs were transfected into cells using Lipofectamine 2000 (Invitrogen).