Cell culture and reagents. Human NSCLC (HCC827, H1299, H23, H820, H1975, PC9, H1573, H1793, H460, and A549) and normal human bronchial epithelial (BEAS-2B) cell lines were purchased from the American Type Culture Collection (ATCC, VA, USA). All cells were grown and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene, Gyeongsangbuk-do, South Korea) at 37°C in a humidified atmosphere containing 5% CO2. RSL3, ferrostatin-1, Z-VAD-FMK, necrostatin-1, and etoposide were purchased from Selleck Chemicals (TX, USA). Chloroquine diphosphate salt, and bafilomycin A1 were purchased from Signma-Aldrich (MO, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich). The oligonucleotide sequences of human GPX4 siRNA (siGPX4) were as follows: 5ʹ-UUCGAUAUGUUCAGCAAGAUU-3ʹ (sense) and 5ʹ-UCUUGCUGAACAUAUCGAAUU-3ʹ (antisense) (Shanghai GenePharma Co. Ltd., Shanghai, China).
Cell viability and proliferation assays. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, MO, USA). Cells were seeded in 96 well culture plates at a density of 1 × 103 per well and treated with the indicated concentrations of erastin, RSL3, ferrostatin-1, Z-VAD-FMK, chloroquine diphosphate salt, bafilomycin A1, and necrostatin-1. After incubation, 10 µL MTT solution (0.5 mg/mL) dissolved in distilled water and 20 µL medium was added to each well. After 2 h of additional incubation, the MTT solution was discarded, and DMSO was added. Absorbance was measured at a wavelength of 570 nm using a microplate reader (SpectraMax Plus 384, CA, USA).
For cell proliferation measurements, cell counting analysis was used. cells were seeded in 60 mm dishes at a density of 2 × 105 per dish. The following day, the cells were treated with either 100 nM RSL3 or 1 µM ferrostatin-1 for 24h. The number of cells were counted using a hematocytometer after trypan blue staining (Sigma-Aldrich, MO, USA). Cells were imaged using an inverted light microscope (Olympus IX71, Olympus Corp, Japan).
RNA isolation and quantitative real-time PCR (qRT-PCR). Total RNA was extracted from cells using TRIzol reagent (Invitrogen, CA, USA). cDNA was synthesized from total RNA using a reverse transcription kit (Lapopass, Cosmo Genetech, Seoul, South Korea) following the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green Q Master (Labopass) and a QuantStudio 3 Real-Time PCR Instrument (Applied Biosystems, MA, USA). The oligonucleotide primers that were used for qRT-PCR are as follows; GPX4 sense: 5ʹ-AGAGATCAAAGAGTTCGCCGC-3ʹ, antisense: 5ʹ-TCTTCATCCACTTCCACA GCG-3ʹ; GAPDH sense: 5ʹ-ACCCACTCCTCCACCTTTGA-3ʹ, antisense: 5ʹ-CTGTTGCTG TAGCCAAATTCGT-3ʹ. The Ct values for GPX4 were normalized to GAPDH. Each variable was assessed in triplicate and repeated in three independent experiments.
Immunoblotting. Cells were lysed in RIPA buffer that contained a protease/phosphatase inhibitor cocktail (Sigma-Aldrich, MO, USA) at 4°C for 30 min and used for immunoblotting. A total of 20 µg of protein was loaded per lane of a SDS-PAGE gel and separated by electrophoresis. The proteins were then transferred onto either nitrocellulose (GE Healthcare, IL, USA) or PVDF membranes (IPVH00010, Millipore, MA, USA). The membranes were subsequently probed with primary antibodies and incubated with either goat anti-mouse IgG (Cell Signaling, 7076, MA, USA) or goat anti-rabbit IgG (Cell Signaling,7074) secondary antibodies conjugated with horseradish peroxidase (HRP). Chemiluminescence was detected using an enhanced chemiluminescence (ECL) system (Translab, South Korea). The following primary antibodies were used: GPX4 (ab125066, Abcam), SCL7A11 (Cell Signaling, 12691, MA, USA), Nrf2 (ab62352, Abcam), Keap1 (Cell Signaling, 8047, MA, USA), HO-1 (Cell Signaling, 5853, MA, USA), p62 (ab56416, Abcam), LC3 (PM036, MBL), and β-actin (Santa Cruz Biotechnology, sc-47778).
Flow cytometry. Cell death was measured using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, NJ, USA) following the manufacturer’s instructions. Cells were seeded at a density of 2 × 105 in 60 mm dishes. The following day, cells were treated with RSL3 (100 nM) and/or ferrostatin-1 (1 µM) for 24 h and then the cells were harvested with culture medium and washed twice with cold PBS. The pellet was resuspended in 200 µL of ice cold binding buffer and labeled with 5 µL FITC annexin V and 5 µL PI for 15 min at RT in the dark. Cell death was analyzed using a flow cytometer (BD Bioscience, BD FACSCanto™ II Flow Cytometry System, NJ, USA).
Labile ferrous ion pool (LIP) analysis. The intracellular labile iron (II) pool (LIP) was measured using a FerroOrange kit (F374; Dojindo, Kumamoto, Japan) following the manufacturer’s instructions. FerroOrange is a fluorescence probe that detects labile iron (II) ions (Fe2+) only. Cells were seeded in 96 well plates at a density of 1 × 104 cells per well and treated with RSL3 and ferrostatin-1 for 24 h. The culture medium was then removed and washed with serum-free RPMI-1640 medium. The cells were incubated with 1 uM FerroOrange in serum-free RPMI-1640 at 37°C for 30 min. FerroOrange intensity was measured at an excitation wavelength of 543 nm and an emission wavelength of 580 nm using an Enspire Multi Plate Reader (PerkinElmer, MA, USA). Each experiment was performed in triplicate and repeated in three independent experiments.
Lipid peroxidation and ROS analysis. Lipid peroxidation activity in cells was analyzed using BODIPY 581/591 C11 (Invitrogen, D3861, MA, USA). For live cell imaging, cells were seeded in a confocal dish at a density of 1 × 104. The following day, the cells were treated with either RSL3 (100 nM) or ferrostatin-1 (1 µM) for 24 h. Next, the cells were incubated in fresh medium with BODIPY 581/591 C11 (2 µM) and covered with a sterile coverslip before incubation at 37°C for 30 min. BODIPY fluorescence was measured at RT using LSM 780 NLO (Carl Zeiss, Baden-Württemberg, Germany). Fluorescence intensity was analysed using the ZEN software (Carl Zeiss, Baden-Württemberg, Germany). Cellular ROS levels were analyzed using DCFA/H2DCFDA-cellular ROS assay kit (ab11385, abcam) after treatment of RSL3 for 6h following the manufacturer’s instructions. The absorbance was measured at an excitation wavelength of 543 nm and an emission wavelength of 580 nm using an Enspire Multi Plate Reader (PerkinElmer, MA, USA). ROS generation of live cells with DCFDA was also measured using LSM 780 NLO (Carl Zeiss, Baden-Württemberg, Germany).
GSH/GSSG analysis. The GSH concentration and GSH/GSSG ratio were calculated using the EZ-Glutathione assay kit (Dogenbio, Seoul, South Korea) following the manufacturer’s instructions. Cells were seeded at a density of 5 × 105 cells per dish in 60 mm dishes. The following day, cells were treated with RSL3 and ferrostatin-1 for 24 h. Cells were harvested and sonicated with PBS, and the cell debris were removed. The absorbance was measured at 412 nm using a microplate reader (SpectraMax Plus 384). Each experiment was performed in triplicate and repeated in three independent experiments.
Autophagic flux assessment. Autophagic flux was assessed in live cells by mRFP-GFP-LC3 expressing plasmid, which kindly given by Professor T. Yoshimori at Department of Genetics, Graduate School of Medicine, Osaka University, Japan43. Cells cultured on confocal dish were transfected with the mRFP-GFP-LC3 plasmid for 24 h, and were treated with or without RSL3 for 24 h. The fluorescence images were obtained with a Laser Scanning Confocal Microscope (LSM800, Carl Zeiss), and visualized with the 63x water immersion objective lenses.
In vivo zebrafish tumor model. Zebrafish (Danio rerio) and embryos were bred and maintained according to standard procedures. All animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation of Sookmyung Women's University (SMWU-IACUC-1712-036-03) and performed as previously described44. Approximately 50 fluorescent cell tracker CM‑Dil-labeled H1299 or H23 cells were injected into the yolk sac of zebrafish embryos at 2 days post-fertilization (dpf). One day post injection (dpi), the zebrafish larvae were treated with RSL3 (100nM) and maintained at 34°C for 5 days. Fluorescence image acquisition was performed using a Zeiss LSM700 confocal microscope (Carl Zeiss AG). The area penetrated by the CM‑Dil-labeled cancer cells was quantified using ImageJ software (version 1.52n, NIH) and normalized to cancer cells in untreated zebrafish embryos for each group.
Statistical Analysis. SPSS statistics version 25 software (IBM, NY, USA) and GraphPad Prism version 5 software (GraphPad, CA, USA) were used for statistical analysis. Experimental results were presented as the mean ± SD from at least two independent experiments. T-tests were performed to compare two independent groups. Comparisons of multiple groups were analyzed using ANOVA. Significance was defined as: # = 0.05 < p (No significance); * = 0.01 < p < 0.05; ** = 0.001 < p < 0.01; *** = p < 0.001.