2.1 Cell culture and treatment

The normal glial cell line HEB (cat. no. HUM-iCell-n009) and human glioblastoma cell lines including A172 (cat. no. iCell-h002), LN229 (cat. no. iCell-h124) and T98G (cat. no. iCell-h210) were supplied from iCell Bioscience Inc (Shanghai, China). All cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% foetal bovine serum (Gibco) and 1% penicillin–streptomycin (Sigma-Aldrich) in a humid incubator at 37 °C with 5% CO2. To explore the mechanism of RBMS1 associated with ferroptosis in regulating GBM cells, ferroptosis inhibitor Fer-1 (1 µM) was used to treat T98G cells for 1 h [15].

2.2 Cell transfection

To reduce RBMS1 expression, short hairpin RNAs targeting RBMS1 (sh-RBMS1-1/2) and the corresponding scrambled sequence as a negative control (sh-NC) were constructed by GenePharma (Shanghai, China). These recombinants were transfected into T98G cells using Lipofectamine 2000 reagent at 37 ℃ for 48 h according to the manufacturer’s instructions. After 48 h, T98G cells were harvested for follow-up experiments.

2.3 Reverse transcription-quantitative PCR (RT-qPCR)

The total RNA that extracted from sample T98G cells with TRizol reagent (Biosharp) was reverse transcribed into cDNA using a commercial RevertAid™ cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Afterwards, the amplification of templates was conducted with SYBR Green PCR Master Mix (Takara, Toyobo, Japan) on the 7500 Fast Real-time PCR system according to the manufacturer’s instructions. 2−ΔΔCt method was used for the calculation of relative gene expression [16]. The following were the primer sequences: RBMS1 forward (F), 5ʹ-TGGCATAGAGAAGGAGAGGCT-3ʹ and reverse (R), 5ʹ-TGAGGGAGTTAACACGGCAC-3ʹ or GAPDH F, 5ʹ-TGTGGGCATCAATGGATTTGG-3ʹ and reverse R, 5ʹ-ACACCATGTATTCCGGGTCAAT-3ʹ.

2.4 Western blot

The concentration of total proteins that extracted from sample T98G cells with RIPA lysis buffer (Solarbio) was quantified using bicinchoninic acid (BCA) protein assay kits (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. The proteins that separated by 8% SDS-PAGE were transferred to PVDF membranes which were sealed by 5% BSA for 2 h at room temperature. Subsequently, the membranes were successively immunoblotted with primary antibodies targeting RBMS1 (ab150353; 1:1000; Abcam), Ki67 (ab92742; 1:5000; Abcam), PCNA (ab92552; 1:1000; Abcam), Bcl-2 (ab182858; 1:2000; Abcam), Bax (ab182733; 1:2000; Abcam), cleaved-caspase3 (ab32042; 1:500; Abcam), caspase3 (ab32351; 1:5000; Abcam), E-Cadherin (ab40772; 1:1000; Abcam), N-Cadherin (ab76011; 1:5000; Abcam), Vimentin (ab92547; 1:1000; Abcam), Snail (ab216347; 1:1000; Abcam), SLC7A11 (ab307601; 1:1000; Abcam), GPX4 (ab125066; 1:1000; Abcam), ACSL4 (ab155282; 1:10,000; Abcam), MMP2 (ab92536; 1:1000; Abcam), MMP9 (ab76003; 1:1000; Abcam) or GAPDH (ab9485; 1:2500; Abcam) overnight at 4 ℃. On the next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721; 1:2000; Abcam) at room temperature for 1 h. Finally, the protein bands were visualized with enhanced chemiluminescence (ECL) detection reagent (Shanghai Yeasen Biotechnology Co., Ltd.) and ImageJ software (version 1.49, National Institutes of Health) was applied for the analysis of protein density.

2.5 Counting Kit-8 assay (CCK-8) assay

The effects of RBMS1 knockdown on the proliferation of T98G cells were investigated through the conduction of CCK-8 assay. Briefly, T98G cells were inoculated into 96-well plates at a density of 3 × 104 cells/well and then incubated at 37 °C for 24 h. After that, each well was added with 10 μL of CCK-8 reagent (Beyotime Institute of Biotechnology) to incubate cells for additional 2 h. Finally, the absorbance at 450 nm was detected with a microplate reader.

2.6 Colony formation assay

T98G cells were initially inoculated into 6-well plates at a density of 1 × 103 cells/well and then incubated for 14 days. After that, the colonies were fixed with 100% methanol for 10 min at room temperature and stained by 0.5% crystal violet solution for 30 min. With the application of a microscope, the colonies were observed.

2.7 Flow cytometry

The collected T98G cells were washed with pre-cold phosphate-buffered saline (PBS) and then got re-suspended in 100 μL binding buffer. After that, T98G cells were incubated with 2 μL Annexin‐V‐FITC (20 μg/mL, Sigma) on ice in the dark for 15 min and subsequently stained by propidium iodide (PI, BD Biosciences). Finally, the apoptotic cells were analyzed utilizing a FACS Calibur Flow cytometer (BD Bioscience) in accordance with the recommend specifications.

2.8 Wound healing

T98G cells were initially inoculated into 6-well plates at a density of 2 × 105 cells/well and then incubated until cell fusion has reached 70–80%. The wounds in the monolayer were made using a 200-µL pipette tip. Following, the PBS-rinsed cells were maintained in serum-free medium for 24 h at 37 °C. Finally, the images of wounds were observed at 0 and 24 h with an inverted microscope (Olympus Corp).

2.9 Transwell assay

T98G cells were initially inoculated into serum-free medium (200 μL) in the upper chambers pre-coated with Matrigel, at 37 °C for 1 h and the medium containing 10% FBS was injected on the lower chambers. Following 24 h of incubation at 37 °C, the invading cells on the lower surface were fixed with 4% paraformaldehyde and stained by 0.1% crystal violet for 30 min. Finally, the images of cells were captured with an inverted microscope.

2.10 Thiobarbituric acid reactive substance (TBARS) assay

For the detection of lipid peroxidation in T98G cells, TBARS Assay Kits (cat. no. E-BC-K298-M; Elabscience) were performed according to the manufacturer’s instructions. Briefly, T98G cells were incubated with 0.5 mL 15% trichloroacetic acid and 10 μL of 500 mM butylated hydroxyanisole (BHA) and then centrifuged at 10,000×g for 10 min at 4 °C [17]. Subsequently, the collected supernatant was exposed to 0.5 mL 0.375% thiobarbituric acid and then boiled for 10 min. Finally, a microplate reader was employed to detect TBARS at 532 nm.

2.11 Lipid ROS detection

For the detection of lipid ROS, a BODIPY 581/591 C11 kit (cat. no. D3861, Thermo Fisher Scientific) was employed. Briefly, T98G cells were inoculated into 6-well plates at a density of 5 × 104 cells/well for 24 h and then exposed to 2 μM C11-BODIPY (581/591) probe according to the manufacturer’s instructions. Following, the cells were visualized with a laser scanning confocal microscope (Olympus) and analyzed using Image J software.

2.12 Measurement of iron level

For the measurement of total iron level, Iron Assay Kit (cat. no. E-BC-K772-M; Elabscience) was performed according to the manufacturer’s instructions. Briefly, T98G cells were homogenized with iron assay buffer on ice and then centrifuged at 16,000×g for 15 min at 4 °C to obtain lysis supernatant. Subsequently, the collected lysis sample (100 μL) were incubated with 5 μL iron reducer for 30 min at 37 °C, following which was the incubation with 100 μL iron probe. Then, the mixture was incubated for 30 min at 37 °C in the dark and a microscope reader was employed for the detection of absorbance at 593 nm.

2.13 Statistical analysis

All experiments were replicated for three times. The collected experimental data were analyzed with GraphPad Prism 8.0 software (GraphPad software, Inc.) and then presented as mean ± standard deviation. For the demonstration of comparisons among multiple groups, one-way analysis of variance with Tukey’s post-hoc test was used. P < 0.05 indicated statistical significance.