Pfu ultra 2 fusion high fidelity polymerase

Casp1^KO and GSDMD^KO cell lines, U937 cell lines expressing WT, p.S208C, p.S242R, p.M694V, p.M680I under the control of a doxycycline-inducible promoter, have been previously described (Lagrange et al., 2018 (link); Magnotti et al., 2019 (link)). p.[V726A], p.[E244K], ΔPYD, ΔPLD, ΔB-Box, ΔCoiled-coil, ΔB30.2 MEFV were generated by mutagenesis of the pENTR1A-3xFlag MEFV using primers presented in Table S3, pfu ultra II Fusion high fidelity polymerase (Agilent) followed by digestion of the parental plasmid using Dpn1 restriction enzyme. The resulting plasmids were validated by sequencing and the mutated MEFV constructs were transferred into the GFP-expressing plasmid pINDUCER21 (Meerbrey et al., 2011 (link)) under the control of a doxycycline-inducible promoter using LR recombinase (Invitrogen). Lentiviral particles were produced in 293T cells using pMD2.G and psPAX2 (from Didier Trono, Addgene plasmids #12259 and #12260), and pINDUCER-21 plasmids. U937 cells were transduced by spinoculation and selected at day 4 post-transduction based on GFP expression on an Aria cell sorter and maintained polyclonal. Pyrin expression was induced by treatment with doxycycline (1 μg.mL⁻¹) for 16 h before stimulation. All parental cell lines were tested for mycoplasma contamination.