Figure 3. Molecular characterization of RAF1-deficient lesions.

(a) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. (b) Immunoblot analysis of signaling pathways in xenograft samples (n=3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in (a). (c) YAP1 expression in the same patient cohort examined in Fig. 1a. Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. (d) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In (a,b), the data are represented as mean±s.e.m., *P≤0.05, **P<0.01, ***P<0.005 according to Student's t-test. (c,d) Middle panels, In the box and whiskers plots (Tukey method), the box represents interquartile range, the middle bar the median, and the whiskers extend to 1.5 times the interquartile range. Statistical analysis was done using Wilcoxon signed rank test; the analysis in the right panels represents the Spearman correlation. r_s and P values are indicated. See also Supplementary Fig. 4.