Mcc950

Mice were sacrificed by cervical dislocation after deep isoflurane-induced anesthesia (4% for induction). Their LGs were cut into small fragments (1 mm³) and treated with MEM-α and 0.1% type 2 collagenase under shaking on a shaker at 50 rpm at 37°C for 30 minutes. After blowing and mixing, as well as centrifuging at 1000 rpm for 5 minutes, the precipitate was uniformly dissolved in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. The supernatant was discarded again, and the resulting precipitate was fully mixed with Dulbecco's modified Eagle's medium containing 20% fetal bovine serum.
We treated the primary LG epithelial cells with MCC950 (10 µM/mL; TargetMol) for 24 hours in order to inhibit the NLRP3 inflammasome. For antioxidation experiments, the primary LG epithelial cells were treated with an N-acetyl-L-cysteine (NAC) antioxidant (1 mM; Sigma, St. Louis, MO, USA) for 24 hours. Thereafter, the treated primary LG epithelial cells of passage 2 were collected for Oil Red O (ORO) staining, JC-1 staining, TUNEL staining, and Western blotting.

MSU, nigericin, ATP, poly(deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic:polycytidylic acid (poly (I:C)), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO), and ultrapure LPS were purchased from Sigma–Aldrich (Munich, Germany). Pam3CSK4 was obtained from InvivoGen (Toulouse, France). MCC950 and CDN were from TargetMol (Boston, MA, USA). Lfn-Flic was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. Anti-mouse CASPASE-1 (1:1000, AG-20B-0042) was bought from Adipogen (San Diego, CA, USA). Anti-human cleaved IL-1β (1:2000, 12242), anti-human CASPASE-1 (1:2000, 4199 S), anti-mouse IL-1β (1:1000, 12507), anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (1:2000, 60004-1-1g) was purchased from Proteintech (Chicago, IL, USA).