Pro il 1β

All culture materials were purchased from Gibco (Grand Island, NY). LY294002 (PI3K/Akt inhibitor) and rapamycin (p70S6K inhibitor) were purchased from Calbiochem (La Jolla, CA). Mouse monoclonal antibodies (mAB) against SREBP-1c, NLRP3, IL-1β, pro-IL-1β, caspase-1, pro-caspase-1, phospho-Akt, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against phospho-p70S6K, p70S6K, phospho-JAK2, and JAK2 were purchased from Cell Signaling Technology (Beverly, MA). SREBP-1c, NLRP3, JAK2 siRNA and control siRNA were purchased from the National RNAi Core Facility in Academic Sinica, Taipei, Taiwan. The integrin β1-siRNA were purchased from Invitrogen (Carlsbad, CA). P. gingivalis lipopolysaccharide (Pg-LPS) was purchased from InvivoGen (San Diego, CA). Other chemicals of reagent grade were obtained from Sigma (St. Louis, MO).

The cells were harvested and lysed using RIPA buffer with protease inhibitor (Thermo Fisher Scientific), and the protein concentrations were determined by the BCA protein assay (Thermo Fisher Scientific). A total of 40 ug of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes as previously described [23 (link)]. Blots were incubated with primary antibodies against NLRP3 (#15101; Cell signaling technology, Danvers, MA, USA), ASC (#67824; Cell signaling technology), pro-IL-1β (sc-32294; 1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), cleaved IL-1β (#83186; Cell signaling technology), pro-CASP1 (#3866; Cell signaling technology), cleaved CASP1 (#89332; Cell signaling technology), and GAPDH (sc-32233; Santa Cruz Biotechnology) at the 1:1,000 dilution, followed by an incubation with the secondary antibody conjugated to HRP for 1 h in room temperature. The bands were visualized using SuperSignal West Pico PLUS Chemiluminescent (Thermo Fisher Scientific) and luminescent image analyzer (ChemiDoc XRS + Systems, Bio-Rad Laboratories) at multiple exposure settings. For quantification, ImageJ software (National Institutes of Health) were used to analyzed the bands intensity.

Luteolin ( > 99% pure) was purchased from DASF Bio-Tech Ltd (Nanjing, P.R.C.). Paclitaxel (Taxol) injection was purchased from Cisen Pharmaceutical Co., Ltd (Shandong, P.R.C.). Primary antibodies for AIM2, cyclin B1, cdc2, and phospho-cdc2 (Tyr15), PCNA, β-actin, Vimentin, E-cadherin, MMP9, p21, and Tetramethylrhodamine (TRITC)-conjugated secondary antibodies were gained from Biogot Biotechnology Co., Ltd (Louis Park, Minnesota, USA). Antibodies against ASC, pro-caspase-1, caspase-1 p10, pro-IL-1β, and IL-1β were all gained from Santa Cruz Biotechnology Inc. (Delaware Ave Santa Cruz, CA, USA). Crystal violet, 4′,6-diamidino-2-phenylindole (DAPI) and Lipo6000™ transfection reagent were purchased from Beyotime Biotechnology (Nanjing, P.R.C.). AIM2 overexpression plasmid and AIM2 siRNA were purchased from Shanghai Sango Biotechnology (Shanghai, P.R.C.). Cell cycle detection kit was obtained from KeyGen Biotechnology Co., Ltd (Nanjing, P.R.C.). SYBR Green Master Mix was obtained from Vazyme Biotech Co., Ltd (Nanjing, P.R.C.). Matrigel was purchased from Becton, Dickinson and Company (NY, USA). Poly(dA:dT) was purchased from InvivoGen (CA, USA).