Polyinosinic polycytidylic acid

C57BL/6 (H-2b MHC) female mice, 8 week old, were purchased from Harlan (Udine, Italy). All animals were housed at the Animal Facility of the Istituto Nazionale Tumori “Pascale” (Naples,Italy). Mice were housed in number of 2–3 per cage and maintained in a conventional facility on a 12 h light:12 h dark cycle (lights on at 7:00 a.m.) in a temperature-controlled room (22 ± 2 °C) and with food and water ad libitum at all times. The experimental protocols were in compliance with the European Communities Council directive (86/609/EEC) and were approved by the Italian Ministry of Health (approval number 835/2016).
Animals (6 for each group) were injected by sub-cutaneous route with 100 µg of each peptide, emulsified with 50 μg of Polyinosinic:polycytidylic acid [poly(I:C); InvivoGen] adjuvant formulated in PBS (200 μl total volume). Immunization was performed twice a week for a total of six administrations. At the time of sacrifice, spleens were resected and processed into single cell suspensions using a gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions for immunological evaluation.

After acclimation, the 160 scallops were randomly divided into four groups of 40 scallops each. Each group of scallop was injected intramuscularly with any of 50 μL phosphate-buffered saline (PBS; 0.14 M NaCl, 3 mM KCl, 10 mM Na₂HPO₄, and 1.5 mM KH₂PO₄, pH 7.4), 50 μL lipopolysaccharide (LPS; 10 mg/mL in PBS; Sigma, USA), 50 μL peptidoglycan (PGN; 10 mg/mL in PBS; Sigma), or 50 μL polyinosinic–polycytidylic acid (poly(I:C); 1.0 mg/mL in PBS; In vivoGen, USA). The gills of three animals from each group were randomly sampled at 0, 3, 6, 12, 24, 48, and 72 h after PBS, LPS, PGN, and poly(I:C) stimulation. In addition, nine types of tissues (gills, hemocytes, feet, striated muscle, smooth muscle, hepatopancreas, gonad, mantle, and kidney) were collected from three untreated scallops to study the tissue distribution of CfIKK3 mRNA.

NCI-H292 cells (ATCC reference number CRL-1848), a human pulmonary mucoepidermoid carcinoma cell line, BEAS-2B cells (ATCC CRL-9609), a SV-40 transformed bronchial epithelial cell line, and A549 (ATCC CCL-185) a cell line from a lung adenocarcinoma, were cultured in RPMI (NCI-H292) or F12/K Nutrient mixture (BEAS-2B and A549) medium supplemented with Glutamax, antibiotics, and 10% de-complemented fetal calf serum (all reagents from Gibco). Cells were incubated at 37°C in a water-jacketed CO₂ incubator. Cells were infected in serum-free medium with either IAV or PAO1. Alternatively, they were stimulated with either h-IL-1β, 5′ triphosphate double stranded RNA (5′ ppp dsRNA at 1.2 μg/ml) (Invivogen), complexed to lipofectamine 2000 (Invitrogen), with polyinosinic-polycytidylic acid (poly IC at 10 μg/ml) (Invivogen), or with combinations thereof.
Cell viability was assessed by measuring Lactate dehydrogenase (LDH) activity in cell lysates and supernatants, using the CytoTox 96 Nonradioactive Cytotoxicity assay (Promega).
Cells were washed twice with ice-cold PBS and lysed in TrisHCl 50 mM, NaCl 150 mM, NP40 1%, Glycerol 3%, EDTA 2 mM, and EGTA 2 mM buffer. After centrifugation (14,000 rpm, 15 min, 4°C) pellets were discarded. Cell supernatants and lysates were then recovered and stored at −80°C until further analysis.

Melatonin was purchased from Nacalai Tesque (Kyoto, Japan). Murine macrophage cell line, RAW264.7, was cultured in RPMI1640 supplemented with 5% FBS. Transfecting (PH)-PLCδ-GFP plasmid (kindly gifted by Dr. Greg Fairn, Dalhausie Univeristy, Canada) into cells was performed with Neon Transfection System (Thermo Fisher Scientific, Massachusetts, USA). For the Neon transfection, RAW 264.7 cells were transfected with the mixture of plasmid DNA and resuspension buffer and then electroporated with the setting of 1680 V, 20 ms, 1 pulse. Cells were transferred to glass coverslip with RPMI1640 supplemented with 10% FBS for 2 days before microscope observation. For optimal mitochondrial condition, cells were used for all experiments after 3 days of cellular passage. For the hypoxia experiment, RAW264.7 cells were pretreated in an anaerobic chamber for 6 h at 36.5 °C (O₂ < 0.1%). The cells were then treated with Melatonin (1 mM) and infected by EMCV for 16 h. Polyinosinic-polycytidylic acid (poly (I:C), LMW) was purchased from InvivoGen (California, USA). Lipofectamine 3000 (Thermo Fisher Scientific) was used for lipofection for poly (I:C). For immunoblotting, anti-mouse IL-1β antibody (rabbit monoclonal, ab234437, Abcam, Cambridge, UK) and anti-pan actin antibody (mouse monoclonal, ACTN05(C4), ab3280, Abcam) were used.