For data from human samples, statistical significance between means was determined by the nonparametric Mann-Whitney U test. Correlation between TGF-β and CXCR4 mRNA levels was determined by the Pearson correlation coefficient. In order to evaluate the relevance of the autocrine stimulation of TGF-β pathway in the acquisition of mesenchymal-like features, we analyzed the phenotype of six different human liver tumor cell lines whose characteristics are detailed in Supporting Table 1. A correlation between the decrease in E-cadherin and cytokeratin-18 (CK-18) expression, characteristics of an epithelial phenotype,

and the appearance of cells expressing vimentin (a mesenchymal intermediate filament) was observed selleck screening library (Fig. 1A). The acquisition of a mesenchymal-like phenotype occurred concomitantly with an increase in the expression of TGFB1 (Fig. 1B) and with nuclear localization of both SMAD2 and SMAD3 (Supporting Fig. 1). Analysis of TGF-β in the culture medium revealed increased amounts of this cytokine in mesenchymal-like versus epithelial cell lines. Furthermore, conditioned medium from mesenchymal-like HCC cells induced higher Smad2 phosphorylation in immortalized mice hepatocytes (Supporting Fig. 1). With the exception of Compound Library chemical structure the HepG2 cells

that show mutations in NRAS and are resistant to TGF-β-induced suppressor effects,[19] the epithelial phenotype correlated with response to TGF-β as a cytostatic factor, whereas cells with a mesenchymal-like phenotype did not arrest

proliferation in the presence of TGF-β (Fig. 3-mercaptopyruvate sulfurtransferase 1C). This behavior confirms a previous classification of these cell lines according to the TGF-β signature[9] (early for PLC/PRF/5 and Huh7; late for SNU449, HLF). Results in Hep3B indicate that these cells represent a transition from an epithelial to a mesenchymal-like phenotype, since they showed decreased expression of E-cadherin and simultaneous expression of epithelial (CK-18) and mesenchymal (vimentin) intermediate filaments (Fig. 1A). Interestingly, this mixed phenotype correlated with a high activation of the TGF-β pathway (Supporting Fig. 1) and lower suppressor response to this cytokine (Fig. 1C). In summary, mesenchymal-like phenotype in HCC cell lines correlates with autocrine stimulation of the TGF-β pathway and resistance to TGF-β-induced suppressor effects. The analysis of the cytoskeleton organization reflected that cells with more mesenchymal phenotype presented F-actin located in stress fibers, whereas the more epithelial ones showed more pericellular distribution (Fig. 2A, left panels). Cells with mesenchymal characteristics showed CXCR4 in an asymmetric distribution in a great percentage of them (Fig. 2A, right panels). HepG2 cells showed homogeneous distribution of CXCR4 with no apparent polarization, whereas in the epithelial Huh7 and PLC/PRF/5 localization of CXCR4 was variable, with some cells showing polarized areas, but a great percentage containing homogeneous intracellular localization (Fig.

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