Reagents and antibodies

Andro was purchased from Tokyo Chemical Industry (Tokyo, Japan) and prepared as a 10 mM stock solution. Final working concentrations ranged from 1 to 100 μM. Cytosine β-D-arabinofuranoside (cytarabine, Ara-C) and vincristine sulfate (VCR) were obtained from Sigma-Aldrich (St. Louis, MO) and used at final concentrations of 40 and 0.1 μM, respectively after preparation in PBS. N-acetyl-L-cysteine (NAC; Funakoshi, Tokyo, Japan), was prepared as a 1 M stock solution in ultrapure water and used at a final concentration of 3 mM. Erastin (Funakoshi) was dissolved in DMSO to yield a 2 mM stock solution and used at a final concentration of 5 μM. The following primary antibodies were used for western blot analysis and immunohistochemistry: cytochrome c (1:2,000; Funakoshi), β-tubulin (1:3,000; Proteintech Japan, Tokyo, Japan), cleaved caspase-3 (1:100; Cell Signaling, Danvers, MA), FACL4 (1:5,000–40,000; Abcam, Cambridge, UK), and GPX4 (1:3,000; Abcam). Horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:3,000; Jackson ImmunoResearch Laboratories, West Grove, PA) were used for western blot analysis. For immunohistochemistry, anti-rabbit and anti-mouse IgG antibodies labeled with peroxidase (Histofine Simple Stain MAX-PO; Nichirei Corp, Tokyo, Japan) were used.

Cell culture

Jurkat cells (EC88042803) and MOLT-4 cells (JCRB9031), both T-cell acute lymphoblastic leukemia (T-ALL) cell lines, were obtained from DS Pharma Biomedical (Osaka, Japan) and the JCRB Cell Bank (Osaka, Japan), respectively. Nalm-6 cells (JCRB1475), a B-cell acute lymphoblastic leukemia (B-ALL) cell line, were also purchased from the JCRB Cell Bank. All cell lines were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio Inc., Kerrville, TX), 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO/Thermo Fisher Scientific, Carlsbad, CA). Cultures were kept at 37 °C in a humidified atmosphere with 5% CO2. All cells were treated with Andro, Ara-C, VCR, or Erastin at the indicated concentrations. NAC was added 1 h prior to Andro, Ara-C, or VCR treatment. Untreated control cells were harvested after 24 h.

Cell viability assay

All cells were seeded into 96-well plates (Becton and Dickinson, Franklin Lakes, NJ) at 2.5 × 104 cells/well in 100 μL of complete medium and treated with Andro (1–100 μM) for 24 h. Cell viability was assessed using an MTT assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s protocol. Absorbance at 550 nm was measured using a BIO-RAD Benchmark microplate reader (Bio-Rad, Hercules, CA).

Measurement of Annexin V expression, ROS generation, and mitochondrial membrane potential

All cells were seeded into 24-well plates (Falcon, Corning Inc., Corning, NY) and treated with Andro, Ara-C, or VCR for 24 h. Apoptosis was evaluated using the Muse Annexin V and Dead Cell Assay Kit (Merck Millipore, Darmstadt, Germany). ROS levels were measured using the Muse Oxidative Stress Kit (Merck Millipore), and mitochondrial membrane potential was assessed using the Muse MitoPotential Kit (Merck Millipore). All assays were conducted according to the manufacturer’s instructions, and data were acquired using a Muse Cell Analyzer (Merck Millipore).

Western blot analysis

Stimulated Jurkat cells were seeded in 6-well plates (Falcon) at a density of 1 × 106 cells in 3 mL and incubated for 24 h with the specified reagents. Western blot analysis was performed according to the standard protocol described elsewhere [20]. Equal amounts (15 μg/lane) of extracted protein were loaded into each well, separated by SDS-PAGE, and protein bands were analyzed by Image J software (National Institutes of Health, Bethesda, MD).

Immunohistochemistry

Immunohistochemistry was performed according to the protocol described in previous studies [21, 22]. The secondary antibody reaction was conducted for 30 min following the manufacturer’s protocol. The intensity of FACL4-positive cells was quantified using Fiji software, an open-source distribution of Image J software. To ensure consistency, all tissue sections were stained simultaneously under the same conditions. Quantitative analysis was performed according to the protocol described in previous studies [23, 24].

Mitochondrial evaluation using transmission electron microscopy

To assess the effects of ferroptosis induction on mitochondria, Jurkat cells were seeded into 6-well plates (Falcon) and cultured for 24 h in the presence of Andro, Erastin, or Andro and NAC. Cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at 4 °C for 16 h and then rinsed three times with 0.1 M PB. Post-fixation was performed with 1% osmium tetroxide in PB for 1 h on ice. Samples were dehydrated in graded ethanol on ice and replaced with QY-1 (Nisshin-EM Co., Ltd., Tokyo, Japan) before embedding in EPON 812 epoxy resin (TAAB Laboratories Equipment Ltd., Reading, UK) and polymerizing at 60 °C for 72 h. Ultrathin sections (80–100 nm) were prepared using an EM UC7 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and mounted on copper grids. The sections were double-stained with uranyl acetate and lead citrate and observed under a JEM1400-Flash transmission electron microscope (TEM, JEOL, Tokyo, Japan). Areas of mitochondria were measured using ImageJ software.

Cell cycle analysis

Jurkat cells were seeded into 24-well plates (Falcon) at a density of 2 × 105 cells/mL and incubated for 24 h after the addition of the indicated reagents. The cell cycle distribution was analyzed using the Muse Cell Cycle Kit (Merck Millipore) and a Muse Cell Analyzer (Merck Millipore) according to the manufacturer’s instructions.

Statistical analysis

All statistical analyses were performed using EZR, a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria), which is a modified version of R Commander designed to include statistical functions commonly used in biostatistics. All results are expressed as mean ± SD of independent experiments. Comparisons between two groups were performed using Student’s t-test, and one-way ANOVA was applied for comparisons among three or more groups. A p-value < 0.05 was considered statistically significant.