Mouse ifn γ

EL4 cells expressing NLRC5-FL, NLRC5-SA or control vector were treated with mouse IFNγ (20 ng/mL, Peprotech, Rocky Hill, NJ, USA) for 24 h or left untreated. To detect the expression of surface markers, cells suspended in PBS containing 2% FCS were stained with fluorochrome-conjugated antibodies obtained from Biolegend (San Diego, CA, USA) or eBiosciences (San Diego, CA, USA) listed in Table 4. The stained cells were analyzed using CytoFLEX (Beckman Coulter, CA, USA) as previously described [36 (link)]. Statistical analysis was carried out using the GraphPad Prism 8 Software (San Diego, CA, USA).

LPS was purchased from Sigma-Aldrich; Pam₂CSK₄, Pam₃CSK₄, poly(A:U),
and poly(I:C) from Invitrogen; mouse IFN-γ from PeproTech; MRT68601,
bosutinib, dasatinib, and ponatinib from Tocris; losmapimod from Biorbyt;
and nilotinib, imatinib, NG-25, and the other compounds from Table S1 were provided by LifeArc. All compounds
are >95% pure by HPLC analysis.

Mouse BMMs were prepared using osteoclast precursors as described previously^{63 (link)}. BMMs were cultured in 24-well plates (7 × 10⁴ cells/well) in the presence of M-CSF (50 ng/ml) in αMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS). The BMMs were further cultured in the presence or absence of GST-RANKL (100 ng/ml), mouse GM-CSF (10 ng/ml; R&D Systems), mouse IL-4 (10 ng/ml; R&D Systems), or mouse IFN-γ (20 ng/ml; Peprotech, Rock Hill, NJ) with M-CSF (50 ng/ml) for 24 h. Total RNA was collected 24 h after stimulation.

Primary C57BL/6 mouse embryonic fibroblasts (MEFs) were prepared from mice at day 14 post coitum. Irgm1^−/−, Irgm3^−/−, Irgm1/Irgm3^−/−, Irgd^−/− MEFs (kindly provided by Greg Taylor) or Irga6^−/−MEFs [42] (link) were immortalized by transfection of pSV3-neo plasmid [64] (link) and pPur (Clontech, Saint-Germain-en-Laye, France) in a ratio 9∶1 using FuGENE HD (Roche, Mannheim, Germany) according to the manufacturer's instructions. After 24 h, cells were put under selection with 3 µg/ml puromycin (Clontech). Primary and transformed MEFs as well as mouse rectal carcinoma CMT-93 cells (ATCC CCL-223) were cultured in DMEM, high glucose (Invitrogen Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom AG, Berlin, Germany), 2 mM L-glutamine, 1 mM sodium pyruvate, 1× MEM non-essential amino acids, 100 U/ml penicillin and 100 mg/ml streptomycin (all PAA, Pasching, Austria). Human foreskin fibroblasts (Hs27; ATCC CRL-1634) were cultured in IMDM, high glucose (Invitrogen Life Technologies) supplemented with 5% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin (PAA). Cells were stimulated with 200 U/ml of mouse IFNγ (PeproTech, Rocky Hill, NJ, USA) for 24 h. For IDO-inhibition, L-tryptophan (W) (Sigma-Aldrich Co., Saint Louis, MO, USA) was added 15 min prior to infection.