Fig. 2
PITX2A/B/C has distinct functions in the tumorigenicity of HCC. A Western blotting analysis confirmed the ectopic expression of PITX2A/B/C in stable PITX2A, 2B, 2C-transfected LO2, MiHA, Hep3B and PLC-8024 cell, respectively. B The mRNA levels of PITX2A/B/C were detected by qRT-PCR using specific primers in stable PITX2A, 2B, 2C-transfected LO2, MiHA, Hep3B and PLC-8024 cell. ΔCtPITX2A = CtPITX2A-CtGAPDH; ΔCtPITX2B = CtPITX2B-CtGAPDH; ΔCtPITX2C = CtPITX2C -CtGAPDH. The higher the ΔCt, the lower expression level of target gene. C Cell proliferation between PITX2A, 2B, 2C-transfected cells and control cells was compared by XTT assay. D Quantitative analyses of foci numbers of each transfected group are shown in bar chart. E Quantitative analyses of colony numbers of each transfected group are shown in bar chart. B-E The results are expressed as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). F Two shRNAs targeting PITX2 (shPITX2–1 and shPITX2–4) could effectively decrease PITX2 expression in protein level detected by western blotting. shCtl was used as negative control. Representative images of excised orthotopic tumor formed by intrahepatic implantation experiment using PITX2A-transfected LO2 or MiHA cells and control cells (G) or shPITX2 and shCtl transfected PLC-8024 cells (H). I Representative images of PCNA IHC staining in orthotopic tumors generated from cells with PITX2 higher expression and their corresponding control cells (400×, magnification)