Bml g150 0050

For doxycycline-inducible expression of NLRP constructs in immortalized mouse macrophages, we used Tet-ON retroviral system (#631188, Clontech). The different NLRP constructs (deletions or chimeras) were subcloned into pRETROX Tre3G plasmid (Clontech) using Bam HI/Eco RI and transfected using Lipofectamine 2000 into the packaging cell line Gryphon Ampho cell line (Alelle Biotechnology, ABP-RVC-10001). Nlrp3^−/− immortalized mouse macrophages stably expressing the Tet-On 3G transactivator (31 (link)) were transduced with different NLRP constructs or empty vector encoding retroviruses for 2 days. Then, positive macrophages were selected with puromycin (6 μg/ml) and G418 (1.5 mg/ml). For experiments, immortalized mouse macrophages were treated for 16 hours with doxycycline (1 μg/ml; Sigma-Aldrich) and ultrapure LPS 0111:B4 (100 ng/ml; InvivoGen) and then stimulated for 1 hour with nigericin (10 μM; Sigma-Aldrich) or TcdB (1 μg/ml; BML-G150-0050, Enzo), for 6 hours with imiquimod (100 μM; tlrl-imqs, InvivoGen), or for 16 hours with MSU crystals (300 μg/ml; ALX-400-047-M002, Enzo) or with transfected lipoteichoic acid (15 μg/ml; tlrl-pslta, InvivoGen). The specific NLRP3 inhibitor MCC950 (10 μM; CP-456773, Sigma-Aldrich) or the caspase-1 inhibitor IV Ac-YVAD-AOM (100 μM; 400015, Calbiochem) was added 30 min before and during the different stimulations.