Fig. 2

(A) Effect of alcohol feeding on tumor formation in Core and NS5A transgenic mice. Upper panel summary of liver tumor incidence among experimental animals: wt, HCV-Core (Core) and HCV-Core+NS5A (Core/NS5A) transgenic mice. Lower panel-the effect of ethanol feeding to wt and transgenic mice on liver tumor development.

(B) TLR4 is required for tumor development in Western diet fed (WD) HCV Tg mice. Upper panel-tumor incidence among wt and transgenic mice fed WD. Lower panel-the effect of WD on wt and Tg mice tumor development.

(C) TLR4-dependent TICs from DEN/Phenobarbital (Pb) and human HCC (non-viral etiology: no HCV) models. Chimeric mice were generated by transplantation of BM from Tlr4+/+ or Tlr4−/− mice into irradiated Tlr4+/+ or Tlr4−/− mice. DEN-Pb-induced tumor incidence is reduced by recipient TLR4 deficiency but not by donor Tlr4 deficiency. (D and E) Effect of alcohol and WD feeding on plasma endotoxin levels in transgenic mouse genotypes. Alcohol WD feeding equally elevated plasma endotoxin levels in all genotype mice. Serum endotoxin levels are elevated equally in both Tlr4+/+ and Tlr4−/− mice fed ethanol or WD (D) or diethylnitrosamine (DEN)/phenobarbital (Pb) treatment (E). Left panel-effect of alcohol feeding on single and double-transgenic animals. Right panel-effect of WD feeding on single and double transgenic animals. Plasma endotoxin levels were measured in wt, HCV-Core, HCV-NS5A and Tlr4−/− double Tg strains of the wt and single Tg animals, as indicated.

(F) TLR4 is induced in the liver tumor samples from the DEN/Phenobarbital HCC models. Activated TLR4 signaling is evident only in HCV Core fed Western diet (WD) as demonstrated by co-immunoprecipitation analysis showing TRAF6 interaction with TAK1. Induction of TLR4 and NANOG were detected in HCV Core Tg livers fed WD or DEN/Phenobarbital-treated mice.

(G) Tlr4 mutant mice fed Western diet for 12 months have less NANOG protein levels.