Sekm 0007

LPS and LTA (5 μg/mL) were each incubated with Crus2 or P58 (32 μM) or PBS (control) for 1 h at room temperature. J774.1 cells were cultured overnight in a 24-well plate (5 × 10⁵ cells/well) and incubated with Crus2/P58-treated LPS/LTA or untreated LPS/LTA for 24 h at 37 °C. The culture supernatant was collected and used to measure IL-1β (EMC001b, Neobioscience, Shenzhen, China), IL-6 (SEKM0007, Solarbio, Beijing, China) and TNF-α (SEKM0034, Solarbio, Beijing, China) by ELISA according to the manufacturer’s instructions.

MEFs and mouse macrophages were seeded on a 24-well plate and transfected with DDX5, METTL3, and YTHDF2 using Lipofectamine 3000 DNA transfection reagents for 24 h, or with siDDX5, siMETTL3, siYTHDF2, and NC siRNA using Lipofectamine RNAi MAX transfection reagent for 48 h. Next, the cells were infected with P. multocida (multiplicity of infection [MoI] = 20), M. pneumoniae (MoI = 1000), and S. aureus (MoI = 20) for 4 h or they were treated with LPS, FSL-1, and Pam3CSK4 for 4 h. The supernatant from each culture was collected to measure the production of IL-6 using a mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit (SEKM-0007; Solarbio, Beijing, China) according to the manufacturer’s instructions. DDX5^+/+ mice, DDX5^+/- mice, Mettl3 cKO mice, and Mettl3 WT mice were infected with P. multocida (5 × 10³ CFU/g) and S. aureus (5 × 10⁵ CFU/g) for 6, or with M. pneumoniae (5 × 10⁵ CFU/g) for 24 and 36 h. After that, mouse serum was collected to measure the production of IL-6 with the same ELISA kit as above. At the same time, mouse lungs were collected to measure the production of IL-6, as detailed above, and TNF-α using a mouse TNF-α ELISA kit (SEKM-0034; Solarbio, Beijing, China).

The levels of IL-6 (SEKM-0007 and SEKH-0013, Solarbio), tumor necrosis factor-α (TNF-α) (SEKM-0034 and SEKH-0047, Solarbio), IL-10 (SEKM-0010 and SEKH-0018, Solarbio), IL-18 (SEKH-0028 and SEKM-0019), and IL-1β (SEKM-0002 and SEKH-0002) in lung tissues and cell cultures were measured according to the instructions.

The lung tissues were centrifuged (2500 xg) for 10 min at 4 °C, and collected the supernatant for the analysis of TNF-α (#SEKM-0034, Solarbio, Beijing, China) and IL-6 (#SEKM-0007, Solarbio, Beijing, China) levels by corresponding ELISA assay kits. All procedures were implemented following the manufacturers’ instructions. And the results of these two indices in lung tissue supernatant were normalized with the protein concentration measured by Coomassie blue staining kit (Beyotime Biotechnology Co., Ltd. Shanghai, China), and the levels were indicated as ng or pg per protein (mg).