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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Gastroenterology. 2014 May 14;147(3):690–701. doi: 10.1053/j.gastro.2014.05.004

Figure 3. Impact of Yap and β-catenin knockdown in HB cells, singly or dually, on β-catenin and Yap target gene expression.

Figure 3

A. A decrease in expression of Yap and its targets like CTGF, Cyr61, Jag1 and survivin, after silencing Yap in HepG2 cells at 24 and 48 hours after transfection. Transfection of β-catenin siRNA also led to decrease in the expression of Yap and some of its targets like Cyr61 and survivin, although no change in expression of CTGF or Jag1 was observed. Dual knockdown of β-catenin and Yap led to a more pronounced impact on Yap targets Cyr61 and survivin. A decrease in expression of β-catenin and its target genes such as Axin2, c-Myc, Cyclin D1 and DKK1 was evident upon β-catenin silencing in HepG2 cells at both 24 and 48 hours after transfection. While, Yap suppression had no impact on expression of β-catenin, it reduced expression of c-Myc and Cyclin D1 but not Axin2 or Dkk1. Dual knockdown of Yap and β-catenin led to a more pronounced decrease in c-Myc and Cyclin D1 expression. All experiments were conducted at least 3 times and in triplicates. Statistical analysis: a- different from Control; b- different from Scrambled siRNA; c- different from Yap siRNA; d- different from β-catenin siRNA. All p values < 0.001.

B. A significant decrease in β-catenin transactivation was observed in a TopFlash reporter assay at 48 hours after silencing of Yap in HepG2 cells (p<0.01). FopFlash –transfected cells showed no luciferase activity.

C. A significant increase in β-catenin transactivation was observed in a TopFlash reporter assay at 48 hours of Yap silencing in Hep3B cells, a human HCC cell (p<0.01) as compared to negative control siRNA transfected cells.

D. A significant decrease in TEAD-luciferase activity was observed in HepG2 cells that were transfected with siRNA against YAP (p<0.01) or β-catenin (p<0.01) for 48 hours as compared to control siRNA.