Fig. 5

PITX2C enhances stemness through up-regulating the key developmental factors in liver progenitor. A ChIP-PCR results showing the binding of PITX2C at the promoter regions of RALYL. B The promoter region of RALYL was fused with firefly luciferase reporter. The luciferase activity was augmented in PITX2C transfected MIHA cells. The values represent the mean ± SD of three independent experiments (*P < 0.05, two-sided Student’s t-test). C Summary bar chart of relative mRNA expression of PITX2 (top) and RALYL (bottom) in the corresponding transfected cells. Parental cell was used as control. D Association of PITX2C expression with RALYL expression in HCC clinical samples (Cohort-1). The relative expression levels of PITX2C or RALYL in HCC clinical samples were evaluated by ΔΔCt _PITX2C/RALYL = (Ct _{PITX2C/RALYL in tumor} – Ct _{GAPDH in tumor})-20 as the PITX2C and RALYL expression was almost absent in non-tumor tissue. The expression of PITX2C or RALYL was defined as presence in HCC cases in which ΔΔCt _PITX2C/RALYL < 0. The P value was calculated using Pearson Chi-square test. E Quantitative analyses of foci numbers (top) or spheroids (bottom) of each transfected group are shown in bar chart. Values indicate the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). Ctrl: control group; PITX2: PITX2C-transfected group; RALYL: RALYL-transfected group; PITX2-RALYL: both PITX2C and RALYL-transfected group; RALYL-shPITX2: RALYL and shPITX2-transfected group. F Western blotting was performed to determine the expression of TGF-β2, NANOG, PI3K, phosphorylated-AKT (p-AKT), p-STAT3, and c-Jun in the indicated cell lysates. G Biological process identified by DAVID gene ontology analysis