Centrifuge tubes

ATCC (Beijing Beina Chuanglian Biotechnology Institute) provided U-87 and U-251 human glioma cell lines, which were then incubated in F12 and DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States), respectively. Both cell lines were kept in a humidified incubator and maintained at 37°C with 5% carbon dioxide. The negative control (NC) and FOXD3-AS1 siRNA (Thermofisher, United States) were transfected into the glioma cells utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s guidelines. The target sequences for FOXD3-AS1 siRNAs were 5′-GTG​TGG​ACA​AAT​CCT​CCA​AGA-3′ (FOXD3-AS1 si 1) and 5′-GAG​GAG​TTC​CGA​GAG​GAA ATA-3′ (FOXD3-AS1 si 2). The target sequences for FOXD3-AS1-overexpression were F: 5′-ATA​CTC​GAG​CGA​ACA​AAG​GGA​CGA​GAG​ACG​C-3′, R: 5′-ATA​GCG​GCC​GCT​CTT​TAA​AGC​CGC​TCC​CTG​G-3′ (FOXD3-AS1 oe 1) and F: 5′-ATA​CTC​GAG​CGA​AGA​GTA​AGA​GCA​GCG​CAC​C-3′, R: 5′-ATA​GCG​GCC​GCC​GGG​AAA​GAG​CAG​GTA​GGA​C-3′ (FOXD3-AS1 oe 2). At the same time, cell culture dishes/plates, and centrifuge tubes were obtained from NEST Biotechnology Co. Ltd (Wuxi, China)

Primary hRECs were purchased from PromoCell (C-12200, Heidelberg, Germany), and all of the experiments were performed using 2-5 passages of hRECs. hRECs were grown in complete endothelial culture medium ECM (ScienCell, 1001) containing 1% endothelial cell growth supplement (ScienCell, 1052), 5% fetal bovine serum (Gibco, A3160802), and 1% penicillin/streptomycin solution (Gibco, 15140-122) at 37°C in a humidified atmosphere of 5% carbon dioxide. Cell culture plates and centrifuge tubes were purchased from NEST Biotechnology Co. Ltd. (Wuxi, China). For high glucose cells, experiments indicated that the amount of D-glucose (MCE, HY-B0389) was added directly in ECM media to obtain a final concentration of 10, 15, 20, or 30 mM, and a hypertonic group (24.5 mM mannitol and 5.5 mM glucose) was added to exclude hyperosmolarity effects. To detect the role of ferroptotic signals in HG-induced endothelial dysfunction, hRECs were treated with 10 μM apoptosis inhibitor tauroursodeoxycholic acid (TUDCA) (MCE, 35807-85-3), 10 μM necrosis inhibitor necrostatin-1 (MCE, 4311-88-0), 10 μM ferroptosis inhibitor ferrostatin-1 (Fer-1) (MCE, HY-100579), and 10 μM pyroptosis inhibitor tetraethylthiuram disulfide (TETD) (MCE, 97-77-8) for 48 h.

All the oligonucleotides in this work were obtained from Huzhou Hippo Biotechnology Co., Ltd. (Huzhou, China), and the oligonucleotides used in this work are listed in Supplementary Table S1. T4 DNA ligase and Phi29 DNA polymerase were purchased from New England BioLabs (Beverly, MA, United States). Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum were purchased from Gibco. Cell culture dishes/plates, round coverslips, and centrifuge tubes were obtained from NEST Biotechnology Co. Ltd. (Wuxi, China). Hoechst 33,342 was purchased from Abbkine Scientific (Wuhan, China). Calcein-AM staining kit was purchased from Solarbio kit (Beijing, China). Annexin V-fluorescein-5-isothiocyanate (AV-FITC)/PI double staining kit was purchased from Elabscience Biotechnology Co., Ltd.