Hepatitis C viral proteins perturb metabolic liver zonation Marie Moreau, Benjamin Rivière, Serena Vegna, Manar Aoun, Christopher Gard, Jeanne Ramos, Eric Assenat, Urszula Hibner  Journal of Hepatology  Volume 62, Issue 2, Pages 278-285 (February 2015) DOI: 10.1016/j.jhep.2014.09.004 Copyright © 2014 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Steatosis is associated with altered tissue pattern of FASN expression. (A) Haematoxylin and eosin staining of paraffin-embedded liver sections from wild type (wt) and FL-N/35 transgenic mice. (B) Fatty acid synthase immunohistochemistry on wt and FL-N/35 livers. Pericentral and periportal regions are indicated. Scale bars: HE staining 500μm, IHC 500μm, 250μm, and 100μm for upper, middle and lower panels, respectively. (C) Quantification of FASN positivity in 30 periportal regions from wt and FL-N/35 livers. Positivity was arbitrarily set at >30% of labelled hepatocytes. (D) FASN localization in human liver biopsies. 7 porto-centrilobular axes were counted on each of the 47 biopsies analysed. Results are presented as %axes displaying periportal (PP), diffuse 1 and diffuse 2 distribution of FASN, as illustrated in Supplementary Fig. 3. Statistical significance was analysed by the χ2 test (∗∗∗p<0.001, ∗∗∗∗p<0.0001). Journal of Hepatology 2015 62, 278-285DOI: (10.1016/j.jhep.2014.09.004) Copyright © 2014 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Glutamine synthetase expression pattern is perturbed in HCV transgenic mice. (A) Protein expression analysed by immunoblotting with anti-GS antibody with tubulin serving as a loading control (B) Immunohistochemical staining of GS expression in wt and FL-N/35 mice. The scale bars are 500μm. (C) Quantification of GS stained areas in livers from 6 transgenic and 6 control animals were analysed by a two-tailed t- test (∗∗∗p<0.0005). Journal of Hepatology 2015 62, 278-285DOI: (10.1016/j.jhep.2014.09.004) Copyright © 2014 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Glutamine synthetase (GS) expression pattern is altered in human liver biopsies. (A) Immunohistochemical staining of GS expression in human liver biopsies. Score 0 corresponds to centrilobular pattern, score 1 shows a limited spread of GS labelling and score 2 corresponds to GS expression spreading across the entire central to ventral axis. (B and C) GS distribution in 50 biopsies from patients described in Table 2. (B) Results of anatomopathological assessment of IHC from HCV-negative (n=6) and genotype 1 HCV-infected patients (n=44) are presented as % lobules displaying centrilobular (score 0), enlarged (score1) and diffuse (score 2) distribution of GS, as illustrated in (A). Statistical significance was analysed by the χ2 test (∗∗∗∗p<0.0001). (C) ImageJ analysis of biopsies analysed in (B) HCV-negative score 0 and score 1 (n=4 and n=2, respectively), HCV-positive score 0, 1 and 2 (n=17, n=15, and n=10, respectively). Statistical analysis was done by a two-tailed t test (∗p<0.05, ∗∗∗p<0.0005, ∗∗∗∗p<0.0001). Journal of Hepatology 2015 62, 278-285DOI: (10.1016/j.jhep.2014.09.004) Copyright © 2014 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Wnt/β-catenin pathway is activated by HCV proteins. (A) RT-qPCR analysis of Wnt4 and Tcf7l2 (a), Axin2 and Gpr49 (b), LRP5, and LRP6 (c) mRNA, normalized to HPRT mRNA in wt and FL-N/35 mice livers. Results were analysed by a two-tailed t test (∗p<0.05, ∗∗p<0.005). (B) RT-qPCR analysis of Wnt4 and Tcf7l2 (a), Axin2 and Gpr49 (b), LRP5, and LRP6 (c) mRNA, normalized to SFRS4 mRNA in HCV negative and HCV positive human liver samples. Results were analysed by a two-tailed t test (∗p<0.05). (This figure appears in colour on the web.) Journal of Hepatology 2015 62, 278-285DOI: (10.1016/j.jhep.2014.09.004) Copyright © 2014 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2015 62, 278-285DOI: (10. 1016/j. jhep. 2014. 09 Copyright © 2014 European Association for the Study of the Liver Terms and Conditions