Fer 1

Erastin (Catalog number S7242, Selleck, TX, USA), a ferroptosis inducer, was diluted with dimethyl sulfoxide (DMSO) to obtain different working solutions and added to the cell medium at different concentrations 12 h before CVB3 infection. Ferrostatin-1 (Fer-1, catalog number S7243, Selleck, TX, USA), an effective lipid ROS scavenger, has been used as a classic ferroptosis inhibitor. Deferoxamine mesylate (DFO, catalog number S5742, Selleck, TX, USA) is a chelator that inhibits ferroptosis. The two inhibitors were diluted in DMSO and used to pretreat cells for 24 h before CVB3 infection at different concentrations.

Immortalized mouse hippocampal HT22 cells were cultured in DMEM (C11995500BT, Gibco, USA) containing 10% FBS and 100 units of penicillin and 100 µg/ml streptomycin. Cells were then incubated at 37°C in a 5% CO₂ atmosphere. For experiments, HT22 cells were pretreated with 12.5 μM Fer-1 (S7243, Selleck, USA) or 1 μM Lipo-1 (S7699, Selleck, USA) for 2 h. Then, 300 μM FAC (F5879, Sigma, USA) was added to the medium for 36 h.

The HT22 cell line (a hippocampal neuronal cell line) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). HT22 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NE, USA) containing fetal bovine serum (FBS; Gibco, Grand Island, NE, USA) and 1% penicillin-streptomycin (PS; Gibco, Grand Island, NE, USA) and maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. HT22 cells were treated with glutamate (10 mM) for 24 h to induce ferroptosis. HT22 cells were inoculated in 6-well plates at a density of 3.5 × 10⁵ cells/well. After 24 h of treatment, they were divided into blank control, glutamate, and glutamate + Ferrostatin-1 (Fer-1) groups. HT22 cells were pretreated with Fer-1 (5 μM; Selleckchem, Houston, TX, USA) for 60 min prior to glutamate treatment.

All doses and methods of administration were based on the protocols of previous experiments (Zhang et al., 2019b (link); Zhang et al., 2019c (link); Kuang et al., 2021 (link)). AS-IV (No. 84687–43-4, purity ≥98%) was obtained from Nanjing spring and autumn biotech Co. Ltd. (Nanjing, China). The injection of AS-IV (20 mg/kg) was carried out intraperitoneally (i.p.) at 30 min after the induction of SAH. Fer-1 was purchased from Selleckchem (Houston, TX, United States). Fer-1 (dosage = 2 mg/kg) were administrated i.p. After 30 min of operation. The rats in the sham group were administrated with an equal amount of normal saline (NS). ML385 (50 pmol/5 μl; AOBIOUS, Gloucester, MA, United States), a specific Nrf2 inhibitor, was diluted in DMSO, and was used for intracerebroventricular (i.c.v.) injection at 24 h before SAH.

Trophoblast cells were donated by Prof. Longfei Xiao from the Beijing University of Agriculture and screened for Mycoplasma contamination using a Mycoplasma staining assay kit (C0296; Beyotime, Shanghai, China). Cells were cultivated in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) in a humidified incubator at 37 °C and 5% CO₂. These cells were treated with erastin (5, 10, 15, 20, 30, and 50 µM) or 10 µM Fer-1 (Selleck, Houston, TX, USA), with 0.2% dimethyl sulfoxide (DMSO) as the negative control.
The circAMN1-overexpressing plasmids (pcDNA3.1 circAMN1), silencing RNA (si-circAMN1), and their negative controls (NC; pcDNA3.1 NC and si-NC) were constructed by GenePharma (Shanghai, China). The miR-205_R-1 mimic/inhibitor and NC were constructed by Sangon Biotech (Shanghai, China). These plasmids and RNA were transfected into trophoblast cells at different concentrations using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s protocol. The RNA sequences are presented in Table S3.

Cells were seeded in 12-well plates 12 h prior to treatment, pretreated with 2 μM Fer-1(S7243, Selleckchem, USA), or 32 μg mL⁻¹ CHDV for 24 h. Detection of cellular ROS was based on the peroxide-dependent oxidation of DCFH-DA (BL714A, Biosharp, China) to form a fluorescent compound named dichlorofluorescein (DCF). An hour after irradiation, cells were washed with PBS and incubated with 10 μM DCFH-DA at 37^oC for 30 min. For lipid peroxidation assay, fresh medium containing 5 μM C11-BODIPY dye (D3861, Invitrogen, USA) was added to each well 24 h after irradiation, and the cells were incubated at 37 °C for 30 min. The cells were washed with PBS and trypsinized to obtain a cell suspension. Cellular ROS and lipid peroxidation levels were immediately analyzed by flow cytometry (Canto, BD Biosciences, USA). The visual image of the DCF and C11-BODIPY fluorescence in cells was taken on fluorescence microscopy.

SC line RSC96 was purchased from the cell bank of the China Academy of Sciences (Shanghai, China). The cell line was cultured in DMEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% pen/strep (Gibco), and incubated at 5% CO₂ and 37°C.
To establish an HG cell model RSC96 was treated with DMEM containing 100 mM glucose for 48 h as previously described [15 (link)]. Cells were cultured to logarithmic phase and grouped as follows: normal control group (DMEM containing 25 mM glucose), high-glucose group (DMEM containing 100 mM glucose), Erastin (Selleckchem, Houston, USA) group (DMEM containing 25 mM glucose and 2 μM Erastin), and the ferroptosis inhibitor ferrostatin-1 (Fer-1) (Selleckchem) group (DMEM containing 100 mM glucose and 10 μM Fer-1). Erastin, as an inducer of cell ferroptosis, was used as a positive control.