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. 2016 May 13;7(26):39026–39043. doi: 10.18632/oncotarget.9346

Figure 3. In vitro Wnt-induced fibrogenesis activates signaling networks that are functional in HCCs.

Figure 3

A. RNA levels of the indicated genes in progenitor HepaRG HCC cells treated with Wnt3a after transfection with two β-catenin targeting siRNAs. B. LGR5 mRNA expression in HepaRG and Huh-7 cells treated with BSA (control), the Hedgehog pathway inducer Purmorphamine, Wnt3a and the Hedgehog inhibitor KAAD-cyclopamine. Tomatidine is a cyclopamine structural analog which does not inhibit the Hedgehog pathway. C. Immunodetection of LGR5 in HepaRG cells after 13 days of incubation with Wnt3a or BSA. LGR5, red (FluoProbes 594); nuclei, blue (DAPI). D. RNA expression of the hepatocyte differentiation marker ALDOB in HepaRG cells transfected with LGR5 siRNA. E. Gene networks generated by Weighted Correlation Network Analysis (WGCNA) [44] using mRNA expression data for the indicated genes in 79 HCCs. Networks with a correlation coefficient threshold ≥0.30 plotted with Cytoscape [45]. Closeness between nodes is proportional to the number of connections. Thickness of links is proportional to the correlation coefficients. F. 3D scatterplots showing the relationships between mRNA expression of the indicated genes in 79 HCCs. ACTA2, smooth muscle actin; COL4A1, procollagen, type IV, alpha 1 chain. Multiple regression coefficients (R) are indicated.