L 15 medium

Cells were seeded at 500 cells/well with 1μg/mL Hoechst3342 (Sigma) in μClear 96 well black plate (Dutscher) in L-15 medium (Sigma) supplemented with 10%FBS. After 24 hours, medium was removed and fresh medium containing DMSO or 20 μmol/L compound 1 was added. Migration was followed by fluorescent microscopy (IN Cell Analyzer 1000, GE Healthcare) during 6 hours. Analyses were performed only on cells tracked during the entire assay. Speed and trajectories were computed with Excel software as previously reported [26 (link)].

The bacteria used in this study were reported previously [41 (link)]. Vibrio anguillarum, Vibrio harveyi, Pseudomona fluorescens, and Edwardsiella tarda were cultured in Luria-Bertani (LB) broth at 28 °C. Streptococcus iniae was cultured in Tryptic Soy Broth (TSB) at 28 °C. Bacillus cereus and Bacillus subtilis were cultured in marine 2216E medium at 28 °C. Escherichia coli, Micrococcus luteus, and Staphylococcus aureus were cultured in LB broth at 37 °C. All bacteria were grown overnight in appropriate media and temperature; the cultures were then diluted 1:100 in fresh medium and cultured to the mid-logarithmic phase. The bacteria were harvested by centrifugation and washed three times with PBS. The Japanese flounder cell line FG–9307 [42 ] was cultured at 24 °C in L-15 medium (Sigma, St Louis, MO, USA) containing 10% FBS (ExCell Bio, Shanghai, China), 100 units/mL penicillin, and 100 µg/mL streptomycin.

Leishmania longipalpis embryonic LL5 cells were grown in L-15 medium (SIGMA - Aldrich) supplemented with 10 % fetal bovine serum (Laborclin), 10 % tryptose and 1 % penicillin/streptomycin (100 U/ml/100 mg/ml), at 29 °C [9 (link)].
Bacteria were grown overnight in LB and yeast in YPAD liquid medium. Escherichia coli (strain DH5α) and Staphylococcus aureus (strain ATCC 23235) were grown at 37 °C, Serratia marcescens isolated from field insects [19 (link)] and Saccharomyces cerevisiae (strain YRG-2) were incubated at 30 °C. Each culture was pelleted, washed with sterile phosphate-buffered saline (PBS) pH 7.4, resuspended in fresh PBS at OD₆₀₀ = 0.5, immediately autoclaved, cooled to room temperature, and used in challenge procedures of LL5 cells.
Leishmania infantum chagasi (MHOM/BR/1974/PP75) obtained from the Leishmania collection of Instituto Oswaldo Cruz was maintained in M199 medium, pH 7.0, supplemented with 10 % fetal bovine serum and collected at exponential growth phase, washed with PBS, and resuspended in fresh PBS at 10⁷ parasites/ml for direct use in challenge procedures of LL5 cells.

Phagocytosis by trout IgM⁺ and IgT⁺ B cells was performed as described previously (8 (link)). Briefly, fluorescent beads (Fluoresbrite Yellow Green Microspheres, 1.0 µm in diameter; Polysciences) in 300 μl L-15 medium (Sigma-Aldrich) were seeded in 24-well plates (Nunc) at a density of 10⁷ beads/well and pelleted by centrifugation at 2,500 g for 5 min. Then trout PBLs or HKLs in 300 μl L-15 medium were added to each well at a cell:bead ratio of 1:10, followed by incubation for 3 h at 17°C. After incubation, cell suspensions were centrifuged (100 g for 10 min at 4°C) over a cushion of 3% (weight/volume) BSA (Thermo Scientific) in PBS supplemented with 4.5% d-glucose (Sigma-Aldrich) to remove the non-ingested beads. The collected cells were stained with anti-trout IgM and anti-trout IgT mAbs as described above, followed by FACS to sort the phagocytic and non-phagocytic IgM⁺ and IgT⁺ B cells using BD FACSAria III (BD Biosciences). Cells were collected and subjected to total RNA isolation and cDNA synthesis as described above. The relative expression levels of AMP genes in the phagocytic and non-phagocytic trout B cells were determined by the Ct method and normalized against the internal control EF-1a using the 2^−ΔΔCt method (34 (link)).

Colon cancer cell lines used in this study were purchased from American Type Culture Collection (Sigma-Aldrich Corp, St Louis, MO, USA). HT-29 cells were cultured with RPMI-1640 medium (Sigma- Aldrich), and SW620 cells were cultured with L-15 medium (Sigma- Aldrich). All media were supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Biowest, Nuaille, France). All of the cell lines were grown in 5% CO2 at 37°C in incubators with 100% humidity.
Considering SW620 cell line was more aggressive, and HT-29 was less aggressive than other colon cancer cell lines [3 (link)], both of them were selected in the experiment. Colon cancer cell lines SW620 and HT29 were cultivated in vitro and divided into six groups according to different treated factors, as shown in Table 9.

Human melanoma cancer cell lines WM983A and WM852 (provided by Dr. Meenhard Herlyn, The Wistar Institute, Philadelphia, PA) were grown in 3:1 Dulbecco's modified Eagle's Medium/Nutrient Mixture F12 ham (DMEM-F12, Sigma) and L-15 Medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units of penicillin, and 100 mg/ml streptomycin (#P433, Sigma), at 37°C with 5% CO₂. To induce human GFAT2 over expression, WM852 cells were transfected with 100 ng of plasmid for GFAT over expression using the transfection reagent FuGENE HD (Promega #E2311).

RVFV strain 35/74 was originally isolated from the liver of a sheep that died during a RVFV outbreak in the Free State province of South Africa in 1974^{21 (link)}. The strain was previously passaged four times in suckling mouse brain and three times in BHK cells. The virus used for IV inoculation of sheep was prepared by a further amplification in BHK-21 cells (ATCC CCL-10) cultured in CO₂-independent medium (CIM, Invitrogen), supplemented with 5% FBS (Bodinco) and 1% Pen/Strep (Invitrogen).
To prepare a virus-spiked blood meal for membrane feeding of mosquitoes, the virus was amplified in Aedes albopictus C6/36 cells (ATCC CRL-1660). To this end, C6/36 cells were inoculated with a multiplicity of infection of 0.005 and cultured at 28 °C in absence of CO₂ in L-15 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 2% Tryptose Phosphate Broth (TPB) and 1% MEM nonessential amino acids solution (MEMneaa). At 4 days post infection, culture medium was harvested, cleared by slow-speed centrifugation and titrated using Vero-E6 cells (ATCC CRL-1586), grown in DMEM supplemented with GlutaMAX, 3% FBS, 1% Pen/Strep and 1% Fungizone (DMEM +) at 37 °C and 5% CO₂. Titers were determined using the Spearman-Kärber algorithm^{22 (link),23} .