Acsl4

Cells were lysed with RIPA (R0020, Solarbio, Beijing) containing Protease and Phosphatase Inhibitor Cocktail (P002, NCM Biotech, Suzhou) and collected for centrifugation. The steps in this laboratory are described in the previous article about Western experiments. Antibodies used are as follows: FAM60A (1:1,000, Novus, H00058516-B02P), GPX4 (1:1,000, Proteintech, 67763), ACSL1 (1:1,000, Proteintech, 13989), ACSL4 (1:1,000, Proteintech, 22401), PPARA (1:1,000, Proteintech, 15540), PPARG (1:1,000, Proteintech, 16643), YY1 (1:1,000, CST, #46395), MST1 (1:1,000, CST, #14946), p-MST1/2 (1:800, Abcam, ab79199), YAP (1:1,000, CST, #14074), p-YAP (1:700, CST, #13008), p-LATS1 (1:1,000, CST, #9157), LATS1/2 (1:1,000, Bethyl Laboratories, Montgomery, TX), and ACTB (1:1,000, Abways, AB0035).

Prior to western blotting, the protein concentration of the samples was determined by the Bradford assay method54 (link). Equal amounts of 20 μg protein per sample were separated by SDS-PAGE on an 8–12% polyacrylamide gel. Proteins were subsequently transferred to PVDF membranes (Immun-Blot PVDF, Bio-Rad, Hercules, CA, USA), and membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at room temperature. The following antibodies were used: ACSL4 (1:1000), ADPR (1:1000), CYP1A2 (1:1000), FTCD (1:1000), FBXO2 (1:300) and UGT2B7 (1:100) from Proteintech (Rosemont, IL, USA); PKM2 (1:1000) and GFPT1 (1:1000) from Cell Signaling (Danvers, MA, USA); PCK2 (1:1000) from Abcam (Cambridge, MA, USA). These primary antibodies were diluted in 5% (w/v) skim milk in TBS-T and incubated with membranes overnight at 4 °C. After washing for five minutes in TBS-T three times, horseradish peroxidase-labelled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used for detection for 1 h at room temperature. Visualization of the immunoreactive proteins was accomplished using enhanced chemiluminescence (SuperSignal West Pico, Thermo, Rockford, IL, USA) followed by exposure to X-ray film (XBT, Carestream, Xiamen, Fujian, China).

Human non-small lung cancer A549 cells, H1299 cells were purchased from American Type Culture Collection (Rockville, MD, USA). A549 cells were grown in Dulbecco Modified Eagle Medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany) and 1% penicillin/streptomycin solution (Capricorn Scientific, Ebsdorfergrund, Germany). H1299 cells were grown in Roswell Park Memorial Institute with l-glutamine media (RPMI, Capricorn Scientific, Ebsdorfergrund, Germany). The cells were maintained in the monolayer at 37 °C in a humidified atmosphere with 5% CO₂.
Ferrostatin-1 (Cat# S7243), RSL3 (Cat# S8155) were purchased from Selleck Chemicals (Houston, USA). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cat# M1415) was purchased from Duchefa Biochemie (Harrlem, Netherlands). PrimeScript RT Master mix (Cat# RR036) and TB Green (Cat# RR430) were purchased from TAKARA (Shiga, Japan). The primary antibodies against GAPDH (Cat# 5174) and SLC7A11 (Cat# 12691) were purchased Cell Signaling Technology (Massachusetts, USA). NRF2 (Cat# 16396), FSP1 (Cat# 20886) and ACSL4 (Cat# 22401) were purchased from Proteintech (Rosemont, USA). 4-hydroxynonenal (4-HNE, Cat# ab46546) was purchased from Abcam (Cambridge, UK).

Total protein was extracted from primary spinal cord neurons using Total Protein Extraction Kit (Solarbio). BCA‐100 Protein Quantitative Analysis Kit (Biocolors, Shanghai, China) was used to estimate the concentration of proteins. Equivalent protein samples (25 μg) were separated by 10% SDS/PAGE. The separated samples were transferred onto nitrocellulose membrane (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 2 h and then incubated with the primary antibodies, PTGS2 (1 : 1000; Proteintech, Wuhan, China), GPX4 (1 : 1000; Proteintech), ACSL4 (1 : 3000; Proteintech), AKT (1 : 1000; Proteintech), p‐AKT (1 : 2000; Proteintech), Nrf2 (1 : 1000; Proteintech), HO‐1 (1 : 1000; Proteintech) or β‐actin (1 : 2000; Proteintech) overnight at 4 °C. After washed with Tris‐buffered saline Tween for several times, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody (1 : 5000; Proteintech) at room temperature for 1 h. The data were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The IVD tissues were decalcified, embedded in paraffin, and cut into 5 m slices after being fixed in 4% paraformaldehyde. The paraffin slices were antigen-repaired with citric acid (pH 6.0), blocked with goat serum, and dewaxed with xylene and gradient ethanol. The sections were then treated at 4 °C for an overnight period with primary antibodies to ACSL4 (1:200, Proteintech, USA), Collagen II (1:200, Novus, USA), Aggrecan (1:500, Servicebio, China), and Piezo1 (1:200, Affinity, USA). The sections were exposed to goat anti-rabbit IgG-HRP secondary antibody for 1 h at room temperature the next day. The DAB Substrate kit (ZLI-9018, ZSGB) was used for detection, and the samples were then counterstained with 1% hematoxylin. Using a microscope (Leica DMI3000B, Germany), pictures were recorded. ImageJ quantified the areas that were positive.

Antibodies against Flag (ShanghaiGenomics), β-actin (Genescript), GPX4 (Abcam), ACSL4 (Proteintech), ERK (CST), p-ERK (CST), NRF2 (Abcam) were purchased commercially.
Full-length cDNA of D2HGDH and IDH1 was amplified by PCR and cloned into indicated pBabe and pQCXIH. Point mutations for IDH1 were generated by site-directed mutagenesis and verified by Sanger sequencing.
AG-120 (CSNpharm), IDH-889 (DC Chemicals), erastin (MedChemExpress, MCE), RSL3 (MCE), Deferoxamine mesylate (MCE), Ferrostatin-1 (Selleck Chemicals), (2 R)-2-Hydroxyglutaric Acid Octyl Ester Sodium Salt, and (2S)-2-Hydroxyglutaric Acid Octyl Ester Sodium Salt (Toronto Research Chemicals) were purchased commercially.