Dmem high glucose complete medium

L929 cells were cultured in DMEM high glucose complete medium containing 10% fetal bovine serum (Gibco, United States) and 1% penicillin and streptomycin (Solarbio, China). L929 cells were cultured in a cell incubator at 37°C and 5% CO₂. The cells were digested and counted when the cells grew to the logarithmic phase. After 200 μL cell suspension was inserted into a 96-well plate (2 mm × 2 mm size). TADM and FBADM were put into the cell suspension, co-cultured with the cells for 24 h, fixed with 2.5% glutaraldehyde solution for 24 h, and lyophilized. The infiltration and growth of cells were observed by scanning electron microscope.

Lentivirus-GFP/Lentivirus-mCherry (Heyuan Biotechnology Co., Ltd., China) was used to transfect HUVECs with green fluorescent (GFP) and red fluorescent mCherry protein markers, respectively. HUVECs were grown in 24-well plates at a density of 7 × 10⁴ cells/well, then added with 500 μl RPMI-1640 complete growth medium and cultured at 37°C, in 5% CO₂ incubator (Heraeus Company, Germany) for 24 h. Next, the culture medium was replaced with DMEM high-glucose complete medium (Gibco BRL, United States) with 5 μg/ml polybrene (Heyuan Biotechnology Co., Ltd., China) and preconfigured virus solution with MOI = 40. Polybrene is a cationic polymer, which can neutralize the electric charge to promote binding between the lentivirus and cell membrane. After 72 h of infection, the infection efficiency was evaluated by a fluorescence microscope (Olympus, Japan); 0.5 μg/ml purinomycin was selected to maintain HUVECs labeled with green fluorescent protein (GFP-HUVECs) or red fluorescent protein (mCherry-HUVECs). In the following experiments, GFP-HUVECs were used to characterize the migration of adjacent VECs, and mCherry-HUVECs to indicate the adhesion of circulating VECs.

The FAM-labeled aptamer EpDT3 was got from Shanghai GenePharma Bio-technology Company (Shanghai, China). Recombinant adenovirus expressing the tumor-suppressor gene PTEN (Ad5-PTEN) or reporter gene LacZ (Ad5-LacZ) was obtained from VGTC Gene Technology (Beijing, China). The COOH-PEG₂₀₀₀-COOH (MW 2057) was purchased got from JENKEM Technology Corporation (Beijing, China). Protein marker was obtained from Thermo Fisher Scientific (Vilnius, Lithuania, EU). DMEM high glucose complete medium and RPMI 1640 medium were from Gibco Life Technologies Corporation (Grand Island, NY). 5-fluorouracil (5-FU) injection was provided by Tianjing Kingyork Group Company (Tianjing, China). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp Biotechnology Company (Beijing, China). All other reagents were purchased from Keyang Corporation (Luzhou, China).

Epithelial cell lines (human lymphatic endothelial cells [HLEC] and human umbilical vein endothelial cells [HUVEC]) and human embryonic kidney cell HEK‐293 were used in this study. HLEC, HUVEC, and HEK‐293 were obtained from NeuronBiotech (Shanghai, China). HLEC and HEK‐293 were cultured in complete DMEM high glucose medium (Gibco, Gaithersburg, BRL, USA) supplemented with 10% FBS (fetal bovine serum)(Hyclone, Logan, UT, USA) and 1% penicillin and streptomycin sulfate (Beyotime Biotechnology Company, Jiangsu, China). HUVEC was cultured in RPMI1640 medium (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin sulfate. Cells were incubated at 37°C with 5% CO² and the medium was changed every 2 or 3 days.

The rat AQP4 siRNA oligonucleotide (5′-GCCAAGTGGAGACAGAAGA-3′) and negative control sequence (5′-TTCTCCGAACGTGTCACGT-3′) were purchased from Gene-Pharma (Suzhou, China). For the gene expression study, the full-length sequence (972 bp) of the rat AQP4 gene was synthesized by PCR and cloned into the LV5 vector at the Not I and Nsi I restriction sites by GENEWIZ Biology Company (Beijing, China).
Astrocytes (1 × 10⁶) were cultured in a 6-well culture plate with complete DMEM/high-glucose medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (PAA, Pasching, Austria) and 1% penicillin/streptomycin in an incubator at 37°C with a humidified atmosphere and 5% CO₂. The complete DMEM medium was replaced by Opti-MEM serum-free medium (Gibco) when the cell fusion level reached more than 80%.
The AQP4-LV5 plasmid or AQP4 siRNA was transfected into the rat astrocytes by using Polybrene Transfection Reagent (Sigma-Aldrich, St. Louis, MO). Briefly, the mixture of AQP4-LV5 plasmid or AQP4 siRNA and polybrene (6 μg/mL) was prepared at a 100:1 ratio and transfected into astrocytes, according to the manufacturer's instructions. After 24 hours, the transfection medium was replaced with complete DMEM, and these cells were used for the subsequent experiments.