Pam3csk4

Common carp PBLs were prepared by density gradient centrifugation with Percoll (Sigma-Aldrich, Germany) according to a previous protocol (27 (link)). Briefly, for the isolation of PBLs, diluted blood was layered on top of 65% Percoll and centrifuged. After 25 min of centrifugation at 800 × g, the cells present on the interface of the gradient were collected and washed three times with PBS. The cells were resuspended in complete L-15 (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). Approximately 10⁷ cells/well were seeded in 24-well plates with 500 μl of complete medium. After recovering overnight at 25°C, drug treatment was performed using poly(I:C) (a synthetic analogue of double-stranded RNA) (5 μg/ml, Sigma-Aldrich), LPS (a component of the outer membranes of gram-negative bacteria) (10 µg/ml, Sigma-Aldrich), peptidoglycan (PGN) (the main PAMP of gram-positive bacteria) (10 μg/ml, Sigma-Aldrich), flagellin (a principal component of bacterial flagella) (10 ng/ml, Sigma-Aldrich), and Pam3CSK4 (a synthetic triacylated lipopeptide that can be recognized by TLR1/2) (10 ng/ml, Invitrogen, USA) at different time points (3, 6, 12, and 24 h) according to the previously described protocols (28 (link)). The cells in the control group were stimulated with the same amounts of PBS (denoted by 0 h).

For preparation of Heat-Killed Yersinia, wild type Y. pseudotuberculosis strains were grown overnight at 26° in Luria-Bertani (LB) broth. Overnight cultures were heat killed at 60° for 30–60 min and aliquots were frozen at −80° until use.
Macrophages were stimulated with HKY (MOI = 50), LPS (Sigma, 0.1 µg/mL or 1 µg/mL) and Pam3CSK4 (Invitrogen, 2 µg/mL), poly(I∶C) (InvivoGen, 50 µg/mL) for the desired time points. Cells were washed 3× with PBS and lysed in 2× SDS Laemmli sample buffer.

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.

Thioglycolate-elicited macrophages were stimulated with NAGase (0.5, 1, 2.5, 5 and 10 μg/mL) for 48 h in the cytokine production assay. Pam3CSK4 (1 μg/mL), LPS (1 μg/mL), and IFN-γ (1.5 ng/mL) (Invitrogen, San Diego, CA, USA) were used as positive controls. IL-6, IL-12p40, TNF-α, and IL-10 levels in the supernatant of peritoneal macrophage cultures from BALB/c and C57BL6 mice were measured by the capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from BD Biosciences (Pharmingen, San Diego, CA, USA). The ELISA procedure was performed according to the manufacturer’s protocol. The quantity of cytokines was determined from a standard curve, using murine recombinants IL-12p40, IL-6, TNF-α, and IL-10.

BMDMs pretreated for 16 h with 100 ng/ml Pam3CSK4 (Invitrogen) were infected with L. monocytogenes at an MOI of 1. At 30 min postinfection, the medium was replaced with media containing 50 µg/ml gentamicin. At 6 h postinfection, supernatants were removed and measured for lactate dehydrogenase activity as previously described (67 (link)) using a BioTek Synergy HT spectrophotometer.