Raw264.7 cells

RAW264.7 cells were purchased from Procell Life Science&Technology (Wuhan, China) and authenticated by STR profiling. The cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Gibco, USA) with 10% fetal bovine serum (Australian origin; Gibco) and 1% penicillin-streptomycin (Gibco, USA) at 37 °C with 5% CO₂.
RAW264.7 macrophages were unstimulated to create M0 macrophages. For M1 macrophage creation, RAW264.7 cells were treated with LPS (10 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) and interferon (IFN)-γ (100 ng/ml; PeproTech) for 24 h. For the AAM, RAW264.7 cells were treated with IL-4 (20 ng/ml; PeproTech) for 24 h.
Apoptosis inducer (20 μM, 24 h), pyroptosis inducers (7 μg/ml, 24 h), necrosis inducer (4 x, 24 h), RSL3 inducer (5 μM, 5 h), and erastin inducer (60 μM, 24 h) were used to treat macrophages (M0, M1, and AAM). For 5 h, AAM was treated with RSL3 (5 μM) in the presence or absence of Fer-1 (400 nM). AAM was treated with the 1488 candidates combined with RSL3 (5 μM) for 5 h. AAM was treated with Lie (1, 5, and 10 μM) combined with RSL3 (5 μM) for 5 h. AAM was treated with RSL3 (5 μM) in the presence or absence of Lie (10 μM) for 5 h. AAM was treated with RSL3 (5 μM) in the presence or absence of Lie (10 μM) or bafilomycin A1 (25 nM) for 5 h.

Primary microglia cultures were prepared as previously described.23, 24 Briefly, cerebral cortical cells from newborn C57BL/6 mice were isolated after a 30‐minutes trypsinization (0.25%) and plated in 75‐cm² culture flasks in DMEM with 10% heat‐inactivated foetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Life Technologies GmbH). The culture medium was changed after 24 hours and then once in every 4 days. Two weeks later, microglia were obtained by mild trypsinization. Purified microglia comprised a cell population in which 95% stained positively with CD11b antibodies. The murine macrophage cell line RAW 264.7 cells were purchased from ATCC (Manassas, VA, USA). Microglia and RAW 264.7 cells were activated by 100 ng/mL IFN‐γ for 24 hours (PeproTech, Rocky Hill, CT, USA).

The RAW 264.7 murine macrophage cells and HL-60 human promyelocytic leukemia cells were purchased from the Korean Cell Line Bank. RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). HL-60 cells were incubated under the same conditions using RPMI 1640 medium instead of DMEM. In order to induce differentiation into the neutrophil-like cells, HL-60 cells were incubated with 1.25% v/v DMSO for 5 days without media change. Lipopolysaccharide, gelatin, fibronectin, type I collagen, laminin, Sudan black, neutral red, and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phaseolin was purchased from Molport (CAS: 13401-40-6, Beacon, NY, USA). Polyclonal antibodies against inducible nitric oxide synthase (iNOS), tubulin, nuclear factor kappa B (NF-κB), inhibitor kappa B (I-κB), glyceraldehyde 3-phosphate dehydrogenas (GAPDH), and lamin A were purchased from Santa Cruz Biotechnology. Polyclonal antibodies against endogenous Ninj1 were obtained from Dr. Kyu-Won Kim (Seoul National University, Seoul, Korea).