Additional file 1 of PITX2C increases the stemness features of hepatocellular carcinoma cells by up-regulating key developmental factors in liver progenitor

Additional file 1: Supplementary Materials and Methods. Supplementary Figure 1. PITX2 selection. (A) Heatmap of the expression profiles of the selected genes which were highly expressed in LP and PH. These genes included the specific genes for LP cells (AFP, GATA3, NPNT, FOXA1, SMAD3, FOXF1, CDX2) and the others encoding nuclear protein which showed a similar expression pattern to LP markers. (B) Among those selected nuclear protein genes, PITX2 was located in the central of gene regulatory network (Pathway Common). (C) Screenshot from SeqMan browser (Lasergene software 7.0) showing the varing 5′ sequences of the full-length of PIT2XA/B/C (PITX2-V1 and V6:PITX2A; PITX2-V2, V4 and V5:PITX2B: PITX2-V3: PITX2C). (D) Western blotting analysis confirmed the protein levels of PITX2 in immortalized liver cells and HCC cell lines. GAPDH was used as a loading control. (E) Representative images of FISH staining of PITX2C (red) in HCC cases with low, moderate and relative high expression levels of PITX2C. DAPI (blue) was used for nuclei counterstaining. (F) Kaplan-Meier overall (left) and disease-free (right) survival curve of two HCC groups in TCGA cohort: PITX2 (+), patients with higher PITX2 expression; PITX2 (−), patients with lower PITX2 expression. Supplementary Figure 2. PITX2A/B/C has distinct function in the tumorigenicity of HCC. Representative images of foci formation assay (A) and colony formation (B) in PITX2A/B/C-transfected cells and control cells. (C) Two shRNAs targeting PITX2 (shPITX2–1 and shPITX2–4) effectively decreased the mRNA level of PITX2 in PLC-8024 and SNU449 detected by qRT-PCR. Non-transfected cells were used as controls. Data are presented as the mean ± SD of 3 independent experiments. (*P < 0.05, **P < 0.01, independent Student’s t-test) (D) The cell proliferation between shPITX2 -transfected cells and control cells was compared by XTT assay. The results are expressed as the mean ± SD of three independent experiments. (*P < 0.05, **P < 0.01, independent Student’s t-test). Representative images (left) and summary bar chart (right) of foci formation assay (E) and colony formation in soft agar assay (F) in shPITX2-transfected and control cells. Values indicate the mean ± SD of 3 independent experiments (*P < 0.05; **P < 0.01; independent Student t test). (G) Orthotopic tumor formation was performed via intrahepatic implantation experiments using PITX2A-transfected cells and control cells or shPITX2-transfected cells and control cells. The final tumor volumes are summarized in the dot chart. Average tumor volume is expressed as the mean ± SD of mice. The P value was calculated using paired Student’s t test. (H) Representative images of excised orthotopic tumor formed by intrahepatic implantation experiment using PITX2A-transfected Hep3B cells and control cells. Supplementary Figure 3. PITX2C promotes cell mobility, self-renewal and chemoresistance of HCC. (A) Representative images (top) and bar chart (bottom) of cell migration and invasion abilities in shPITX2-transfected and control cells by Transwell and Matrigel invasion assays. Migrated and invaded cells were stained with crystal violet and counted under a microscope. Values indicate the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01; independent Student t test). (B) Representative images of spheroid formation assay using shPITX2-transfected cells and control cells (left). The numbers of primary and secondary spheroids are calculated in the bar chart (right). Values indicate the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). The apoptotic indexes of PITX2C-transfected (C), shPITX2-transfected cells (D) and control cells were detected by fluorescence-activated cell sorting-based Annexin V/AAD double staining after treatment with 5-Fu or Sorafenib at the indicated concentrations for 48 h. (E) The mRNA levels of AFP and Lgr5 were compared by ΔCt in PITX2C or shPITX2- transfected cells and control cells (ΔCtAFP = CtAFP-CtGAPDH; ΔCtLgr5 = CtLgr5-CtGAPDH). Supplementary Figure 4. Representatives of IHC staining images with anti-EPCAM, CD133, c-Myc and NANOG in tumors induced by 8024-Ctrl, 8024-PITX2C cells with 5-FU treatment. Red arrows indicate cancer stem cells. Supplementary Figure 5. Analysis of the ChIP sequencing data. (A) PITX2C shared similar binding motifs with several key transcription factors in LP. (B) Screenshot from the WashU epigenome browser showing PITX2C binding sites at the promoter of HNF1A, HNF4A, FOXA1, SMAD3, and ARID5B. (C) Heatmap of the expression profile for HNF4A, FOXA1, SMAD and ARID5B in the four stages (ES, EN, LP, PH) of in vitro hepatocyte differentiation model. (D) The expression of PITX2 is positively correlated with that of Wnt5α in GEPIA. Table S1. List of PCR primers for PITX2A/B/C expression.