Endothelial cell growth factor

Human aortic endothelial cells acquired from ATCC (Manassas, VA, USA) were maintained in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% endothelial cell growth factor (ScienCell, Carlsbad, CA, USA), and 1% penicillin-streptomycin (Hyclone). Endothelial cells were cultivated at 37°C in an atmosphere of 5% CO₂. To simulate endothelial cell dysfunction in vitro, endothelial cells were exposed to 100 µg/ml ox-LDL (Sigma, St. Louis, MO, USA) for 24 h, as described previously [23 (link)].

Human colonic adenocarcinoma HT-29 cell lines were provided by the Institute of Cell and Biochemistry, Chinese Academy of Sciences (Shanghai, China), and grown in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS; Gibco, USA) in an incubator (Forma Scientific, USA) at 37 °C under a mixture of 95 % air and 5 % CO₂.
Human lymphatic endothelial cells were primary HDLECs purchased from ScienCell Research Laboratories, USA. Cells were identified by immunefluorescent cytochemical technique via CD31, Podoplanin and LYVE-1, and grown in endothelial cell growth medium (ECGM) with endothelial cell growth factor (ScienCell Research Laboratories) in an incubator (Forma Scientific) with 5 % CO₂ at 37 °C as described previously [23 ], then were used in the experiments at fifth generation of the cells.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), endothelial cell medium (ECM), fetal bovine serum (FBS), penicillin/streptomycin, endothelial cell growth factor, and trypsin neutralization solution were purchased from ScienCell (San Diego, California, USA). 0.25% trypsin-EDTA was purchased from Gibco (Grand Island, NY, USA). 2′,7′-Dichlorofluorescein diacetate DCF-DA was purchased from Beyotime. Primary antibodies Nrf2, HO-1, NQO1, GAPDH, and Lamin B1 were from Proteintech (Wuhan, China). Secondary antibodies including anti-mouse and anti-rabbit were purchased from Bioworld Technology (Minnesota, USA). Bardoxolone and ML385 were purchased from MedChemExpress (Shanghai, China). The specific Nrf2 small-interfering RNA (siRNA) and negative control of siRNA were purchased from GenePharma (Shanghai, China). Enhanced chemiluminescence (ECL) western blotting detection solution was obtained from Beyotime (Shanghai, China).

Human umbilical vein endothelial HUVEC-T1 cells (Procell Life Science & Technology, China), RAW264.7-T1 Mφ (Procell Life Science & Technology, China), oxidized low-density lipoprotein (ox-LDL; Yiyuan Biotechnology, China), tumor necrosis factor-α (TNF-α; Bioss, China), and Transwell chambers (Corning, USA) were used. FITC-labelled anti-human CD44 flow cytometry antibody (Proteintech, USA), high-glucose DMEM (ScienCell, China), CCK-8 kit (Tongren Chemical, Japan), fetal bovine serum (FBS; ScienCell, USA, Israel), and double antibody (Gibco, USA) were purchased for this study. Endothelial cell basal medium (ECM; ScienCell, USA), FBS (ScienCell, USA), endothelial cell growth factor (ScienCell, USA), double antibodies (ScienCell, USA), and Fluorescence microscope (LEICA CTR6000, Germany); laser confocal microscope (Leica SP8, Germany); flow cytometer (Beckman, USA); laser nanoparticle size potential analyzer (Malvern, UK) were used.

Using the conventional culture method (i.e., 37°C, 5% CO₂ and 100% relative humidity in an incubator), the HUVECs were incubated in endothelial culture medium containing 5% fetal bovine serum and 1% endothelial cell growth factor (ScienCell), and the 293Ts were incubated in high-glucose Dulbecco’s modified eagle’s medium (DMEM, HyClone) and placed in saturated humidity culture conditions. The 293Ts were incubated in high-glucose DMEM containing 5% fetal bovine serum (HyClone) until they reached 80%–90% confluence. Then, the 293Ts were cultured for 48 h in high-glucose DMEM containing 2% BSA, and the supernatant was collected.