Corn oil

C16 was prepared as described earlier [5 (link)]. A tribromoethanol injectable solution (Avertin) was prepared as described in Section E in S1 File. In-house prepared Milli-Q water was used. Cremophor EL (Sigma, Germany), corn oil (Wako, Japan), dimethyl sulfoxide (DMSO, Sigma-Aldrich, Germany) anhydrous N,N-dimethylacetamide (DMA, Wako, Japan), ethanol (Merck, Germany), Medigel Sucralose (2 oz cups, ClearH2O, USA), 2-methyl-2-butanol (amylene hydrate, Sigma-Aldrich, Germany) 1,2-propanediol (propylene glycol, PG) (Wako, Japan), polyethylene glycol (PEG) 400 (Sigma, U.S.A), PEG 600 (Alfa Aesar, Great Britain or Sigma, USA), sodium heparin (5000 units/mL injectable solution, LEO Pharma, Belgium) Solutol HS-15 (BASF, Germany) 2,2,2-tribromoethanol (Sigma-Aldrich, Germany) were used as obtained from the indicated suppliers.

C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan) and maintained on a 12-h light/dark cycle with free access to a standard chow diet. Liver fibrosis was induced by repeated intraperitoneal injection of CCl₄ (Wako Pure Chemical, Osaka, Japan), twice a week for 4 weeks. CCl₄ was diluted to 20% in corn oil (Wako) and injected into mice at a dosage of 1 mL/kg. Livers were harvested 3 d after the final CCl₄ injection.

Liver fibrosis was induced by intraperitoneal (i.p.) injections of CCl₄ (0.5 μl/g body weight; Wako) diluted 1:4 in corn oil (Wako) twice a week for 6 weeks as described previously (14 (link)). In brief, CCl₄ is converted into several radical forms by liver cytochrome P450 enzymes. The resulting radicals induce hepatotoxic damage and result in fibrosis. Mice were sacrificed 3 days after the final CCl₄ injection. Liver fibrosis was also induced by feeding choline-deficient L-amino acid-defined (CDAA) diet for 8 weeks (Dyets Inc, Bethlehem, PA) (15 (link)). In brief, CDAA diet affects lipid metabolisms, resulting in steatohepatitis and liver fibrosis (16 (link)). For Treg-cell depletion experiments, FDG mice were injected i.p. with diphtheria toxin (DT; 1 μg in 100 μl PBS; Merck) every other day for 2 weeks (Figure 2A and Supplementary Figure 1A).

Fetal bovine serum (FBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT), the protease inhibitor cocktail, the phosphatase inhibitor cocktail, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, Triton X-100, PEITC, allyl isothiocyanate (AITC), fluorescein isothiocyanate (FITC), phenyl isothiocyanate (PITC), and Nonidet P-40 (NP-40) were purchased from Nacalai Tesque (Kyoto, Japan). Sodium orthovanadate (Na₃VO₄) and corn oil were purchased from Wako Pure Chemical Industries (Osaka Japan). Benzyl isothiocyanate (BITC) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). All of the other reagents used in the study were of analytical grade and obtained from commercial sources.

Male VNUT-deficient (Slc17a9^−/−) mice25 (link)26 (link), and their wild-type littermates, and C57BL/6 mice (Clea, Japan) were used. Male and female Slc17a9-floxed mice (B6.129-Slc17a9^tm1.1Rpa/J), Cx3cr1-Cre^ERT2 transgenic mice (B6.129P2(C)-Cx3cr1^{tm2.1(cre/ERT2)Jung}/J), Gfap-Cre transgenic mice (B6.Cg-Tg(Gfap-cre)73.12Mvs/J) and Rosa26-tdTomato reporter mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) were purchased from Jackson Laboratory (USA). Cd11b-Cre transgenic mice (B6.Cg-Tg(Itgam-cre)^2781Gkl/Flmg)51 (link) were kindly provided by Prof. George Kollias (Biomedical Sciences Research Centre ‘Alexander Fleming'). For induction of Cre recombinase, 4–6-week-old Cx3cr1-Cre^ERT2 mice were injected s.c. with 2 mg tamoxifen (Sigma, USA) solved in 100 μl corn oil (Wako, Japan) once a day for 2 days. All mice used were aged 8–15 weeks at the start of each experiment, and were housed individually and in groups of two or three per cage at a temperature of 22±1 °C with a 12-h light–dark cycle, and were fed food and water ad libitum. Genotyping was performed as previously described. All experimental procedures were performed under the guidelines of Kyushu University.

For induction of Cre recombinase activity, 4–6-week-old Cx3cr1^CreERT2 mice were injected subcutaneously with 2 mg tamoxifen (TAM) (Sigma) dissolved in corn oil (Wako), twice at approximately 48 hours intervals.⁵² (link) To avoid the influence of circulating monocytic cells, we performed the PNI procedure 4–5 weeks after TAM administration.²⁷ (link)