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.

附加文件1：补充材料与方法。补充图1：PITX2筛选。(A) 在LP和PH中高表达的候选基因的表达谱热图。这些基因包括LP细胞特异性基因（AFP、GATA3、NPNT、FOXA1、SMAD3、FOXF1、CDX2），以及与LP标志物表达模式相似的核编码蛋白基因。(B) 在上述筛选出的核蛋白编码基因中，PITX2位于基因调控网络（Pathway Common）的核心位置。(C) SeqMan浏览器（Lasergene软件7.0）截图，展示PITX2A/B/C全长的5′端可变序列：PITX2-V1、V6对应PITX2A；PITX2-V2、V4、V5对应PITX2B；PITX2-V3对应PITX2C。(D) 蛋白质印迹分析验证了永生化肝细胞及肝癌细胞系中PITX2的蛋白水平，以GAPDH作为内参加载对照。(E) 不同PITX2C表达水平（低、中、相对高表达）的肝癌病例中，PITX2C的荧光原位杂交（FISH）代表性染色图像，以DAPI（蓝色）进行细胞核复染。(F) TCGA队列中两组肝癌患者的卡普兰-迈耶总生存期（左）和无病生存期（右）曲线：PITX2(+)为PITX2高表达患者；PITX2(−)为PITX2低表达患者。补充图2：PITX2A/B/C在肝癌致瘤性中具有不同功能。(A)(B) 分别为转染PITX2A/B/C的细胞与对照细胞的灶形成实验代表性图像和平板克隆形成实验图像。(C) 针对PITX2的两条短发夹RNA（shPITX2–1和shPITX2–4）可有效降低PLC-8024和SNU449细胞中PITX2的mRNA水平，经实时定量聚合酶链反应（qRT-PCR）验证，以未转染细胞作为对照。数据以3次独立实验的均值±标准差表示（*P < 0.05，**P < 0.01，独立样本t检验）。(D) 通过XTT实验比较转染shPITX2的细胞与对照细胞的增殖能力，结果以3次独立实验的均值±标准差表示（*P < 0.05，**P < 0.01，独立样本t检验）。(E)(F) 分别为转染shPITX2的细胞与对照细胞的灶形成实验代表性图像（左）及汇总柱状图（右）、软琼脂克隆形成实验汇总柱状图。数值以3次独立实验的均值±标准差表示（*P < 0.05；**P < 0.01；独立样本t检验）。(G) 分别采用转染PITX2A的细胞与对照细胞、转染shPITX2的细胞与对照细胞进行肝内原位移植实验，记录原位成瘤情况，最终肿瘤体积以散点图汇总。小鼠平均肿瘤体积以均值±标准差表示，采用配对t检验计算P值。(H) 采用转染PITX2A的Hep3B细胞与对照细胞进行肝内原位移植实验，切除的原位肿瘤的代表性图像。补充图3：PITX2C促进肝癌细胞的迁移能力、自我更新能力及化疗耐药性。(A) 通过Transwell实验及Matrigel侵袭实验检测转染shPITX2的细胞与对照细胞的迁移和侵袭能力，迁移/侵袭细胞经结晶紫染色后在显微镜下计数，附代表性图像（上）及柱状图（下）。数值以3次独立实验的均值±标准差表示（*P < 0.05；**P < 0.01，独立样本t检验）。(B) 采用转染shPITX2的细胞与对照细胞进行球体形成实验，附代表性图像（左），并统计原代及继代球体数量（右柱状图）。数值以3次独立实验的均值±标准差表示（*P < 0.05，**P < 0.01，独立样本t检验）。(C)(D) 分别采用不同浓度的5-氟尿嘧啶（5-Fu）或索拉非尼（Sorafenib）处理转染PITX2C的细胞、转染shPITX2的细胞及对照细胞48小时后，通过基于荧光激活细胞分选的Annexin V/AAD双染色法检测细胞凋亡指数。(E) 通过ΔCt法比较转染PITX2C或shPITX2的细胞与对照细胞中AFP和Lgr5的mRNA水平（ΔCtAFP = CtAFP - CtGAPDH；ΔCtLgr5 = CtLgr5 - CtGAPDH）。补充图4：经5-FU处理的8024-Ctrl及8024-PITX2C细胞诱导形成的肿瘤中，抗EPCAM、CD133、c-Myc及NANOG的免疫组织化学（IHC）代表性染色图像。红色箭头指示肿瘤干细胞。补充图5：染色质免疫共沉淀测序（ChIP-seq）数据分析。(A) PITX2C与LP中多个关键转录因子具有相似的结合基序。(B) WashU表观基因组浏览器截图，展示PITX2C在HNF1A、HNF4A、FOXA1、SMAD3及ARID5B启动子区域的结合位点。(C) 体外肝细胞分化模型四个阶段（ES、EN、LP、PH）中，HNF4A、FOXA1、SMAD及ARID5B的表达谱热图。(D) 在GEPIA数据库中，PITX2的表达与Wnt5α的表达呈正相关。表S1：PITX2A/B/C表达的PCR引物列表。