Fig. 6
H3K27 acetylation in PITX2C promoter region and activation of the Wnt pathway by PITX2. A Relative expression of Wnt5α/β detected by qPCR in transfected cells compared with control cells (*P < 0.05, **P < 0.01, Student’s t test). B The Wnt5α/β secretion level in the cell culture medium was confirmed by western blotting. Total proteins staining with Coomassie Brilliant Blue were used as a loading control. C Western blotting was performed to determine the expression of key components of the Wnt pathway (Wnt5α/β, c-Met, Frizzled, p-GSK3b, GSK3b, LEF, c-JUN, c-Myc and CD44) in cell lysates from PLC-8024 and MiHA cells. β-Actin was used as a loading control. D Detection of PITX2C expression in LO2 and MiHA cells by qRT-PCR after demethylation (5-Aza-dC) and histone acetylation (trichostatin A) treatment. PITX2C expression can be detected when ΔCtPITX2C (referred to CtPITX2C-CtGAPDH) < 30. E The enrichment of H3K27ac at the promoter regions of PITX2C was determined by ChIP-PCR with anti-H3K27ac antibody in LO2, MiHA, PLC-8024 and 449 cells. F The diagram on the left shows the dynamic expression of key markers and transcription factors during liver development and HCC progression. The diagram on the right illustrates the proposed mechanism of PITX2C upregulation in HCC differentiation and progression