Mcc950 sodium

C57BL/6 wild-type (Shanghai JSJ Laboratory Animal Co, Ltd.) or CRAMP^−/− (Jackson Laboratory) female mice (7–8 weeks old) were used for the lung infection model. S. aureus strains were grown to mid-logarithmic phase, washed once and then resuspended with sterile PBS at 5 × 10⁶ CFU/μl. Mice were anesthetized with 2,2,2-tribromoethanol (3.75–5 mg /25 g), and then 40 μl inoculum was pipetted into the nares of the anesthetized mice slowly. The mice were euthanized 48 h after infection, and their lungs were dissected out. The left lung was homogenized in 0.5 ml of TSB, the homogenized tissue was diluted and plated on TSB agar for CFU determination. The right lung was fixed in 4% formalin and tissue sections were prepared for HE staining and immunohistochemical staining. The remaining right lungs were used for RNA sequencing. For the fatal pneumonia model, 50 mg/kg MCC950 sodium (NLRP3 inflammasome inhibitor, Selleck) or sterile PBS was injected intraperitoneally into mice 2 h before infection by 10⁹ CFU (2 × 10⁷ CFU/μl, 50 μl) S. aureus. Lung tissues were taken out immediately after the mouse died.

As we previously described [13 (link)], neonatal rat ventricular myocytes (NRVMs) were obtained from isolated heart tissues of young Wistar rats. Briefly, the dissected hearts minced in HEPES-buffered nic acid saline solution. Non-myocyte contaminants were removed by two rounds of pre-plating for 1.5 h on 100-mm plastic cell culture dishes under a culture condition of 37 °C and 5% CO₂. Then, the cardiomyocytes were seeded into different culture dishes with medium containing serum. After incubation for 24 hours, medium without serum was used to replace the serum-containing medium.
The normal H9c2 cardiomyocyte cell line was purchased from American Type Culture Collection (ATCC). Cells were cultured with 1640 medium containing 10% fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37 °C. Actinomycin D was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used at the concentration of 5 μg/ml. MCC950 sodium (CP-456773, CRID3 sodium salt), a NLRP3 inhibitor, was used at the concentration of 7.0 nM in cell culture and 12 mg/kg in animal treatment (Selleck, Shanghai, China). For osmotic stress, hyperosmotic medium was made by adding various concentrations of D-sorbitol (Sigma-Aldrich) to regular 1640 medium for different durations. Standard 1640 culture condition was used as isosmotic media.

Male C57BL/6J mice (2 months old, weighing 20–25 g) were purchased from the Experimental Animal Center of Anhui Medical University [SCXK (Wan) 2016-0009]. The mice were kept in a room with constant temperature (22 ± 2°C), appropriate relative humidity (45–65%), and well-ventilated conditions. A 12 h light/dark cycle was maintained, and the mice were allowed free access to food and water. The mice were adaptively fed for one week before the experiments. The experimental procedures were approved by the Ethics Committee of Anhui University of Chinese Medicine, Hefei, China (Approval No. AHUCM-mouse-20210205).
Calpeptin was purchased from Selleckchem (S7396, HPLC > 97%, USA). N-Acetyl-L-cysteine (NAC) was purchased from Beyotime Biotechnology (S0077, HPLC > 97%, Shanghai, China). MCC950 sodium, a selective NLRP3 inflammasome inhibitor, was purchased from Selleckchem (S7809, HPLC > 99.57%).