Hcmec

Human cardiac microvascular endothelial cells (HCMECs) (ScienCell Research Laboratories, California, USA) were maintained in complete ECM, which included 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS), and 1% penicillin/ streptomycin (P/S), and incubated in a 37°C, 5% CO2 incubator according to the manufacturer's instructions. When the confluency reached 90%, cells were washed with DPBS, digested with 0.25% trypsin-EDTA, and subcultured at a ratio of 1:3 in a Petri dish precoated with 4mL Bovine Plasma Fibronectin (BPF) (ScienCell Research Laboratories).
HCMECs were cultured in normoxic conditions to 30-35% confluency and then divided into 5 groups: (i) control group: HCMECs were cultured under normoxic conditions; (ii) hypoxia group: HCMECs were cultured under hypoxia conditions (1% O2, 5% CO2, and 94% N2) (Thermo Fisher Scientific) for 3 days to establish hypoxia-induced EndMT [28 (link)]; (iii) hypoxia + high-dose TXL group: HCMECs were pretreated with TXL (600 ug·ml⁻¹) for 4 h and cultured under hypoxia conditions for 3 days; (iv) hypoxia + middle-dose TXL group: HCMECs were pretreated with TXL (400 ug·ml⁻¹) for 4 h and cultured under hypoxia conditions for 3 days; (v) hypoxia + low-dose TXL group: HCMECs were pretreated with TXL(200 ug·ml⁻¹) for 4 h and cultured under hypoxia conditions for 3 days.

Human Cerebral Microvascular Endothelial Cell (hCMEC) were purchased from Cellutions Biosystems Inc.(Ontario, Canada) and were passed in endothelial cell growth medium EndoGRO-MV Complete Culture Media Kit (Millipore) supplemented with human basic fibroblast growth factor (Sigma) and penicillin-streptomycin (Life technologies) [47 (link)]. To generate stable IRS-1-overexpressing hCMEC, a vector pTagRFE-N harboring IRS-1cDNA was transfected into 5 × 10⁵ hCMEC cells using lipofectamine 2000 reagent (Invitrogen) at 80% confluence for 48 h. Different clones with IRS-1 overexpression were isolated after G418 selection (500ug/mL, Gibco) of the transfected cells over two weeks, and validated by immunoblotting.