Luzindole

The RAW264.7 macrophage cell line was sourced from the American Type Culture Collection (ATCC), located in Manassas, VA, USA. These cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco brand) which was fortified with 10% fetal bovine serum (Gibco) and an additional 1% penicillin/streptomycin antibiotic blend (Gibco). The culture conditions maintained were at a steady temperature of 37°C under a humidified atmosphere containing 5% CO₂. RAW264.7 cells were seeded onto 10 cm cell culture dishes (Corning Inc., New York, USA) and the medium was refreshed every two days to ensure optimal growth. Cell passage was carried out once the cells achieved approximately 90% confluence. This process involved discarding the used culture medium, followed by rinsing the adherent cells twice with PBS. Subsequently, the cells were detached and re-suspended in fresh medium at a ratio of 1:3 for further propagation. In subsequent experiments, RAW264.7 cells were treated with 1 μg/ml LPS (L3024, Sigma, MO, USA) for 24 h as described in our previous publication [59 (link)]. Erastin (10 μM) (Selleck, USA), rapamycin (20 μM) (Selleck), 4-P-PDOT (10 μM) (Sigma) and luzindole (10 μM) (Sigma) were treated 3 h before LPS stimulation. MT (500 μM) (Sigma) was treated 1 h after LPS stimulation. All cells were verified to be free of mycoplasma.

Melatonin, ferric ammonium citrate (FAC) and luzindole were purchased from Sigma (St, Louis. Mo. USA). Melatonin, FAC and luzindole were dissolved in dimethyl sulfoxide (DMSO) and the final culture concentration of DMSO was ≤ 0.1%. Other chemicals were all purchased from Sigma (St, Louis. Mo. USA). Stem cell medium was purchased from Stem Cell (Canada). Calcein-AM (40719ES60) was purchased from Shanghai YEASEN Biotechnology. Trypan blue (T-6146) was purchased from Sigma (St, Louis. Mo. USA). Live/Dead Kit (1736918) was purchased from Life technologies. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was purchased from Roche Company (Roche, Germany). PI/Hoechst staining (CA1120) was purchased from Solarbio (Beijing Solarbio Science & Technology, China).

MC3TC-E1 murine preosteoblast cells were cultured in α-Minimum Essential Medium (α-MEM, Gibco BRL, Gaithersburg, MD, USA) added with 10% heat-inactivated fetal bovine serum (FBS, Gibco BRL) and 1% Penicillin-Streptomycin (PS) at 37 °C with 5% CO₂ in a humidified incubator. To experimentally simulate microgravity with the 3D clinostat, cells were cultured in T-25 cell culture flasks (Falcon, Lincoln Park, NJ, USA) completely filled with a 1:1 mixture of α-MEM and Leibovitz’s L-15 (Invitrogen, Auckland, NZ, USA) supplemented with 10% FBS and 1% PS, with or without melatonin (Sigma-Aldrich, St. Louis, MO, USA) and/or luzindole, melatonin receptor antagonist, (20 µM) (Sigma-Aldrich) and sealed. The culture dishes were completely filled in order to eliminate any possible effects of air bubbles and shear forces.

All experimental protocols had been approved by the Clinical Research Ethics Committee of the Peking Union Medical College Hospital. NSCs were obtained from 13.5‐day‐old embryos of Wistar rats using an established method 36. In brief, the telencephalon was separated under a stereotaxic microscope and dissected into small pieces. NSCs were released by incubation of these dissected brain tissues with 0.25% trypsin and kept in neurobasal medium (Hyclone, Logan, UT, USA) with 2% B27 (Invitrogen, USA), basic fibroblast growth factor (bFGF; Invitrogen, Carlsbad, CA, USA), epidermal growth factor (Invitrogen) and their inhibitor (TrkB‐Fc and MAB212, respectively) at 37°C under a humidified atmosphere containing 5% CO₂. Melatonin, IL‐18 and luzindole were obtained from Sigma‐Aldrich. Cells were treated with or without IL‐18 (1–100 ng/ml) in the presence of Melatonin (10 ng/ml) and/or luzindole (5 μM). All mouse experiments are carried out in accordance with the relevant institutional and national guidelines and regulations, approved by the Animal Care and Use Committee of Peking Union Medical College Hospital and conform to the relevant regulatory standards.

Cultures were treated with lipopolysaccharide (LPS) (1 µg/mL) and ATP (5 mM) (Sigma-Aldrich, Madrid, Spain) to create a model of NLRP3 inflammasome activation. Pharmacological treatments were melatonin (10 nM), α-bungarotoxin (α-bgt, 100 nM) and luzindole (1 µM) (Sigma-Aldrich, Madrid, Spain). The compounds used to assess autophagy were chloroquine (inhibitor; Sigma-Aldrich, Madrid, Spain) and rapamycin (inductor; Sigma-Aldrich, Madrid, Spain).