The mouse macrophage cell lines RAW 264.7, and MH-s were purchased from American Type Culture Collection (Manassas, VA, USA). Another mouse macrophage cell line J774.1 was from RIKEN BRC (Tsukuba, Japan). Cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) supplemented with 10% fatal calf serum and 1% Penicillin-Streptomycin (Gibco5,000 U/mL, ThermoFisher Scientific). We conducted the experiments with cell with early passage after thawing the vials. All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2.

The cells (5.0 × 105 cells in 1 ml per well) were seeded on 12-well cell culture plates. Twenty-four hours later cells were stimulated with each silica particle (100 μg/ml). This dose was determined based on the result of preliminary dose-response experiment (Supplementary Fig. 15). In the experiment to assess ROS involvement, an ROS inhibitor (NAC; final concentration 10 mM), or gp91ds-tat (final concentration 20 μM) was added simultaneously with silica particles. Six hours later, the cell-culture dishes were washed with PBS and 1 ml TRIzol (Life Technologies Corp., Carlsbad, CA, USA) was added to isolate total RNA. Isolation of RNA was conducted according to the manufacturer’s protocol, and quantified using NanoDrop ND-1000 (Thermo Fisher Scientific) Only samples with A230/260 above 1.6 were used for subsequent analysis. For gene expression assessments, 3–5 independent experiments were conducted with biological replication.

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