Ferrostatin 1

Antibodies against GAPDH (#97166S, 1 : 1000), PARP (#9542S, 1 : 1000), caspase-3 (#9662S, 1 : 1000), cleaved caspase-3 (#9664S, 1 : 1000), caspase-8 (#9746S, 1 : 1000), cleaved caspase-8 (#9496S, 1 : 1000), Bax (#5023S, 1 : 1000), Bcl-2 (#15071S, 1 : 1000), p-RIP1 (Ser166; #65746S, 1 : 1000), RIP3 (#95702S, 1 : 1000), anti-rabbit IgG, HRP-linked antibody (#7074S, 1 : 3000), and anti-mouse IgG, HRP-linked antibody (#7076S, 1 : 3000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MLKL (#ab184718, 1 : 1000), p-MLKL (Ser358; #ab187091, 1 : 1000), RIP1 (#ab72139, 1 : 1000), and p-RIP3 (Ser227; #ab209384, 1 : 1000) were purchased from Abcam (Cambridge, UK). Cladribine, homoharringtonine, Z-VAD-FMK, cycloheximide, chloroquine, ferrostatin-1, and necrostatin-1 were purchased from TargetMol (Shanghai, China). Aclarubicin was obtained from APExBIO (Houston, TX). A stock solution containing cladribine, homoharringtonine, and aclarubicin was prepared in DMSO and kept at -80°C. TNF-alpha was purchased from ABclonal (Wuhan, China).

Ferroptosis inhibitors including Ferrostatin-1 (T6500; CAS: 347174-05-4), α-Vitamin E (T1648; CAS: 59-02-9) and Deferasirox (T1457; CAS: 201530-41-8) were purchased from TargetMol, China. Fer-1 and DFX were dissolved in dimethyl sulfoxide (DMSO) solution at the concentration of 10 mM while Vit E was dissolved in DMSO at the concentration of 100 mM. IL-1β was obtained from Sigma, Uited States (SRP3083). Human primary chondrocytes together with the culture system were bought from the Beijing Beina Chuanglian Institute of Biotechnology. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C incubator under condition of 5% CO₂. After reaching 80%-90% confluence, cells were digested with trypsin (Gibco, United States) and passaged twice per week at a split ratio of 1:3. Cells at passages 3-4 were used for the subsequent experiments.