Il 4 404 ml

MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin in an atmosphere of 5% CO2, and 95% air at 37℃. According to the previous report [18 ], MH-S cells were polarized as M1 or M2 macrophages with the indicated stimulants: 50 ng/mL LPS (L3024, Sigma, Mo) 24 h for M1 polarization, and 10 ng/mL IL-4 (404-ML, R&D Systems) 24 h for M2 polarization. The 3-DZNeP (Selleckchem, Houston, TX, USA) (0, 1, 2, 5 and 10 μM) was added 24 h before LPS or IL-4 stimulation. The small interfering (si) RNA oligonucleotides targeted to EZH2 (GenePharma, Shanghai, PR China) were used to knock down EZH2 more specifically in vitro. The MH-S cells were transfected with EZH2 siRNA (100 pmol) with lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) or scrambled siRNA (100 pmol) according to the manufacturer’s instructions 24 h before LPS or IL-4 stimulation. After different stimulations, cells and supernatants were harvested for analyses. All of the in vitro experiments were repeated at least three times.

GW9662 (#70785) and PD146176 (#10010518) were purchased from Cayman Chemical. GW4869 (#6823-69-4) was purchased from Sigma-Aldrich. TGF-β1 (240-B-010) and IL-4 (404-ML) were purchased from R&D Systems. 15-HETE and 15d-PGJ₂ were purchased from Enzo Life Sciences. Lipoxin A4 was purchased from Neogene. Mouse IgG Blocking Reagent (MKB-2213) was purchased from Vector Laboratories.

MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), and Raw264.7 cells were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin in an atmosphere of 5% CO₂ and 95% air at 37°C. According to the previous report [16 (link)], MH-S and Raw264.7 cells were polarized as M1 or M2 macrophages with the indicated stimulants: 50 ng/ml LPS (L3024, Sigma, Mo) 24 h for M1 polarization and 10 ng/ml IL-4 (404-ML, R&D Systems) 24 h for M2 polarization. CircN4bp1 overexpressed vector (pc-circN4bp1, 2 μg), circN4bp1 silence vectors (sh-circN4bp1, 2 μg), miR-138-5p mimics (50 pmol), miR-138-5p inhibitor (50 pmol), and the corresponding control vectors were constructed by GenePharma (Shanghai, China). MH-S cells and Raw264.7 cells were transfected with the above vectors by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions 24 h before LPS or IL-4 stimulation. Stably transfected cells are finally verified by quantitative real-time PCR. After different stimulations, cells and supernatants were harvested for analyses. All the in vitro experiments were repeated at least three times.