Cell culture

LN229 and T98G cell lines were obtained from American Type Culture Collection (ATCC) and maintained in DMEM culture medium (BasalMedia, China) containing 5% and 10% fetal bovine serum and 1% penicillin/streptomycin, respectively. The culture temperature was 37 °C, and the CO2 concentration was 5%. The cells came from our laboratory stocks and, after resuscitation, these cells were used for no more than 10 generations.

Antibodies and reagents

Juglone, Chloroquine (CQ-1), Ferrostatain-1 (Fer-1), Liproxstatin-1 (Lip-1), Mdivi-1 (Mdivi), Necrostatin-1 (Nec-1) and Z-VAD-FMK (Z-VAD) were purchased from Selleck. 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFDA) were purchased from MCE. Antibodies against ACSL4, PTGS2, GPX4, Ki67, 4HNE were purchased from Abcam. Antibodies against XCT, FTH1, p38, pp38, JNK, p-JNK, GSK3α/β, p-GSK3α/β, MMp2 and MMp9 were purchased from Cell Signal Technology. Antibodies against TFRC were purchased from Invitrogen. Antibodies against α-Tubulin and GAPDH, as well as secondary antibodies, were purchased from Hua-an Biotechnology. Details and information of antibodies are provided in Additional file 1: Table S1.

Cell viability assay

Approximately 3000–5000 cells were inoculated into each well of a 96-well plate. After 24 h of treatment with different concentrations of juglone with or without inhibitor, the supernatant was discarded, 100 μL of DMEM medium containing 10% CCK-8 (APE × BIO, K1018) was added to each well, incubated for 1 h at 37 °C, then the absorbance was measured at a wavelength of 450 nm.

Colony formation assessment

Approximately 1500–2000 cells per well were inoculated in a 6-well plate. Until the cells grew into visible colonies. Then LN229 or T98G cell lines were maintained in juglone medium for 24 h (cells pre-treated with inhibitor for 1 h), then the medium was replaced with normal growth medium. The same operation needed to be repeated approximately 2 times, with the cells maintained for about 15 days. Fixed with 4% polyformaldehyde for 30 min, stained with crystal violet solution (1% crystal violet in 95% ethanol) for 2 h.

Western blotting analysis

RIPA buffer supplemented with protease inhibitors was used to extract total protein; protein quantitation was conducted using the BCA protein assay kit (Biosharp, China). Samples were electrophoresed on a 10% SDS polyacrylamide gel (SDS-PAGE), then were transferred onto an NC membrane. The membrane was then blocked with 5% non-fat milk for 1 h, followed by washing, incubation with primary and secondary antibodies, and finally detected with enhanced chemiluminescence. The Nuclear and Cytoplasmic Protein Extraction Kit (78833, Thermo Scientific) was used for nuclear Nrf2 and cytosol Nrf2 extraction according to the manufacturer’s instructions, GAPDH and histone H3 (1:3000; Abcam) were served as internal reference proteins.

Immunofluorescence assay

LN229 and T98G cells treated with different concentrations of Juglone for 24 h were fixed with 4% polyformaldehyde for 15 min. After fixation, the cells were washed twice with PBS and treated with PBS solution of 0.2% Triton-X 100 for 15 min. Then, the cells were incubated with 5% skim milk powder at room temperature for 1 h. Primary antibody was incubated at 4 °C for 12 h, and the secondary antibody was incubated at room temperature for 1 h, DAPI staining was done for 10 min, followed by the addition of anti-fluorescence quenching agents and observed with a fluorescence detection microscope. Images were captured and analyzed subsequently with the MetaView software.

Immunoprecipitation assay

After the cells were treated with different concentrations of juglone for 24 h, the cells were collected, lysed with a lysis buffer to obtain the supernatant, and centrifuged to obtain the supernatant, followed by overnight incubation at 4 °C with 2 μL of the antibody and 20 μL of the Protein A/G beads (Thermo Scientific). The sample was then centrifuged, washed three times with IP buffer, then the resulting protein sample was resuspended in 1 × loading buffer and subjected to a metal bath at 100 °C for 10 min, and the immunoprecipitated protein was analyzed by immunoblotting with anti-ubiquitin antibody.

Intracellular reactive oxygen species (ROS) determination

Approximately 2 × 105 cells were seeded onto 6-well plates. After 24 h of incubation, LN229 and T98 cells were treated with different methods for another 24 h, then replaced with HBSS containing 10 μmol/L DCFDA for incubation for 30 min. The cells were washed twice with PBS to remove DCFDA. Then the cells were resuspended in 500 μL HBSS and detected by flow cytometry, and the results are analyzed using FlowJo V10 software. Meanwhile, DCFDA was also used for immunofluorescence detection. LN229 and T98 cells were seeded in 6-well plate and subjected to different treatments for 24 h. Then they were stained with 10 μmol/L DCFDA for 1 h and then stained with Hoechst for 10 min. The fluorescence microscope was used to analyse the results.

Glutathione (GSH) and malonaldehyde (MDA) levels were measured

The measurements of intracellular GSH and MDA levels were determined using GSH test kit (G263, Tong Ren, Japan) and MDA test kit (Sangon, China) respectively. All experimental operations were completed according to the kit operating instructions.

Potential targets prediction and screening of juglone

The ferroptosis-related genes were sourced from the FerrDb database (http://www.zhounan.org/ferrdb) [36]. The potential targets of juglone were predicted using Swiss Target Prediction (http://www.swisstargetprediction.ch/) by applying the downloaded Juglone’s SDF and mol2 format [37]. The protein–protein interaction (PPI) network was obtained from the STRING database 11.0 (http://string-db.org/) and was analyzed and visualized through Cytoscape 2.8.2 [38]. Further, the R language software was used to analyze GO and KEGG data and draw volcano plot and heatmaps.

Molecular docking was performed to predict the interaction pattern

The crystal structure corresponding to the Keap1 and p38 proteins was obtained from the RCSB PDB database (Structural Bioinformatics Protein Data Bank) [39]. The protein crystals obtained were processed separately using the Protein Preparation Wizard module in Schrödinger software. The 2D structure data file of the compound juglone was processed using the LigPrep module in Schrödinger to generate all its 3D chiral conformations. The prepared ligand compound Juglone was docked with the active sites of the p38 and Keap1 protein structures, respectively, and the scores were calculated.

Construction of overexpression plasmid

Nrf2 overexpression vector was purchased from Genepharma (Shanghai, China). Overexpression plasmid (pcDNA3.1-Nrf2) was transfected into logarithmically growing LN229 and T98G cells using Lipofectamine 3000 (Thermo Fisher Scientific). After transfection, cells were collected and the increased expression level of Nrf2 was confirmed by western blot assays.

Transfection of interfering RNA (siRNA)

The si-RNA targeting Nrf2 and its negative control siRNA (si-control) were obtained from GenePharma (Shanghai, China). The sequences of the siRNAs are shown in Additional file 1: Table S2. Transfection was conducted according to Lipofectamine 3000 (Thermo Fisher Scientific) instructions.

The xenograft tumor model was performed

LN229 cell suspension (4 × 106 cells/100 μL) was subcutaneously injected into the axilla of 6-week-old nude mice (Sibeifu Biotechnology Co., Ltd, Beijing, China). When the tumor volume was visible (7 days after inoculation), all animals were randomly divided into four groups, each with 6 mice (3 females, 3 males), which were the control group (saline), juglone group (200 mg/kg/day), Fer-1 (0.8 mg/kg/day) group, and Fer-1 (0.8 mg/kg/day) + juglone (200 mg/kg/day) group, receiving abdominal injections every other day (3 times a week), with tumor size measured every 3 days, one mouse from each group died early during experimental procedure. 17 days after the treatment, the mice were euthanized and the tumors were removed. Then, subsequent Western blot analysis and immunohistochemical experiments were conducted with tumor tissues obtained from different pharmaceutical groups. This animal experiment was approved by the Ethical Committee of China Medical University (Approval No.: CMUXN2023016; Approval Date: June 28, 2023).

Data analysis

Statistical analyses were performed using GraphPad Prism9 (GraphPad software). All experiments were performed at least three independent times and results were expressed as the mean ± SD (standard deviation). p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).