Wortmannin

Eight- to ten-week-old male C57BL/6 J mice purchased from Harlan Laboratories (Indianapolis, IN, USA) were adapted to the environment for one week before the study. The inducible liver IR knockout (iLIRKO) mice were described previously^{21 (link)}. Fifteen-week-old male mice were studied 2 weeks after 3 consecutive injections with tamoxifen (1.5 mg/mouse). All mice were housed in colony cages with a 12 h/12 h light/dark cycle in a temperature-controlled environment (lights off at 3:00 pm). Mice had free access to water and regular diet (65% carbohydrate, 11% fat and 24% protein). For “fasting” and “refed” conditions, mice were either fasted for 24 h, or refed overnight on a regular diet with 20% glucose in tap water after 24 h fasting. For insulin signaling inhibitors experiments, mice were injected twice (18 hours and 2 hours) before sacrifice with wortmannin (2 mg/kg, InVivoGen, CA, USA) or rapamycin (4.5 mg/kg, InVivoGen). Livers were frozen in liquid nitrogen and kept at −80 °C until use. Plasma samples were frozen and stored at −80 °C until use. All procedures were carried out according to the French guidelines for the care and use of experimental animals. All animal studies were approved by the “Direction départementale des services vétérinaires de Paris”.

Lymphocytes are the cell type most often used when investigating DNA damage [36 (link)]. Especially the human TK6 lymphocyte cell line has been widely utilized in genotoxicity studies [37 (link), 38 (link)]. For this reason, we used TK6 (ATCC CRL-8015) cells, a p53-competent, human lymphoblast cell line. Cells were cultured in Roswell Park Memorial Medium without phenol red (RPMI1640; PanBioTech) supplemented with 10% fetal bovine serum, 2% glutamine, and 1% penicillin/streptomycin (all Sigma). All incubations were performed in cell culture conditions (CB210; Binder) at 37°C, 95% humidity, and 5% carbon dioxide. As ROS scavengers, catalase (cat; 20 μg/ml), glutathione (GSH; 1 mM), or superoxide dismutase (SOD; 100 U/ml) was used (all Sigma). As enzyme or signaling inhibitors, Z-VAD-FMK (R&D Biosciences), SB202190 (Sigma), KU55933 (SelleckChem), Ly294002 (Cell Signaling Technologies), wortmannin (InvivoGen), or SP600125 (Santa Cruz Biotechnology) was used at different concentrations and incubated with cells for 1 h prior ROS or UV treatment. Final concentrations for a selected choice of inhibitors were 1 μM for KU55933, 1 μM for SB202190, and 25 μM for Z-VAD-FMK.

Sendai virus (SeV) Cantell and Z strains were grown in chicken eggs 11 days after fertilisation. Influenza virus PR8 strain was grown in the lungs of BALB/c mice. Betanodavirus was used for medaka infection experiments. Titres of SeV and influenza viruses were measured by infecting macaque monkey kidney-derived LLC-MK2 cells, and Madin–Darby canine kidney cells, respectively. Anti-optineurin polyclonal (Cayman, #100000), anti-optineurin monoclonal (Santa Cruz Biotechnology, sc-166576), anti-LC3 monoclonal (MBL, M186-3), anti-GFAP polyclonal (Abcam, ab7260), anti-Iba1 polyclonal (Wako, #019–19741), anti-neurofilament monoclonal (Covance, SMI32), anti-actin monoclonal (Chemicon, MAB1501), horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG polyclonal (Abcam, ab98799), and HRP-conjugated donkey anti-rabbit IgG polyclonal (Abcam, ab97085) antibodies were used for western blotting and immunocytochemistry. The autophagy inhibitor, wortmannin (InvivoGen), was added to culture media at the indicated concentrations. This study was approved by the Hiroshima University biosafety committee for living modified organisms (approved number: 2022-316-2).