C/EBPα inhibits hepatocellular carcinoma by reducing Notch3/Hes1/p27 cascades  Yi-Chao Shi, Hong Zhao, Chuan Yin, Xin Zeng, Qing Zhang, Wen-Ping Xu, Ji Wei, Fei Chen, Wei-Fen Xie  Digestive and Liver Disease  Volume 45, Issue 10, Pages 844-851 (October 2013) DOI: 10.1016/j.dld.2013.03.013 Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 1 Overexpression of C/EBPα reduces the tumourigenesis of hepatoma cells in vitro. (A) Cell Proliferation Assay for Huh7 cells treated with AdC/EBPα or AdGFP. A significant suppressing effect in Huh7 hepatoma cells was observed on the sixth day after infection with AdC/EBPα compared with those infected with AdGFP or the control group. Three independent experiments were performed and the data represent mean±SD. *P<0.05 (compared to AdGFP or untreated controls). (B) Colony formation ability was detected after virus infection for two weeks. Representative images for three independent experiments are shown. (C) Flow cytometric analysis of Huh7 cells infected with AdC/EBPα or control AdGFP was taken on the fourth day. (D) The number of cells in G0/G1 and S phases determined by flow cytometric analysis. Bars are mean±SD (n=3). (E and F) The proliferation inhibition rates of human cancer cells isolated from two independent samples (LIX006 and LIX010) or tumour associated fibroblasts isolated from two independent samples (L100908B and L100825) treated with AdGFP or AdC/EBPα and determined by Cell Counting Kit-8 assay. At least three independent experiments were performed (mean±SD). Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 2 Overexpression of C/EBPα inhibits subcutaneous HCC xenograft growth in mice. (A) Established subcutaneous xenografts of Huh7 were intratumourally treated with AdC/EBPα or the control AdGFP three times per week for two weeks. (B, C) Tumour growth was assessed by serial measurements in AdC/EBPα-treated or AdGFP-treated mice (n=5). Mean tumour volumes before and after three weeks of treatment and statistical comparisons are illustrated in the bar graph. Error bars indicate standard deviation from the mean. *P<0.01 (compared to AdGFP). (D) The final tumour weight from AdC/EBPα-treated or AdGFP-treated mice was analyzed at sacrifice (n=5). *P<0.05 (compared to AdGFP). HCC: hepatocellular carcinoma. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 3 Overexpression of C/EBPα suppresses the growth of orthotopic hepatocellular carcinoma xenograft in mice. (A) Orthotopic xenograft model were established in nude mice by direct injection of Huh7 cells into liver in situ. The liver of the mice were collected within three weeks after administration of C/EBP and the control AdGFP. Tumour nodules are indicated with yellow dashed line. (B) Liver tumour weight was analyzed within three weeks after administration of AdC/EBP or the control AdGFP (n=5). *P<0.05 (compared to AdGFP). (C) Sections from formalin fixed orthotopic liver tumour tissues from AdC/EBPα-treatment or AdGFP-treatment mice were stained by immunohistochemistry for C/EBPα, PCNA and Ki67 (magnification 400×). Examples of strongly positive-stained cells are indicated by black arrows. Scale bars: 50μm. PCNA: proliferating cell nuclear antigen. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 4 Overexpression C/EBPα inhibited the activation of Notch3 pathway. (A) Real-time PCR analysis of Notch3 expression in Huh7 cells after being infected with AdC/EBPα or the control AdGFP. Values represent mean±SD (n=3). **P<0.01 (compared to AdGFP). (B) Whole cell lysates were subjected to Western blot analysis to detect the expression of Notch3 protein after infection with AdC/EBPα or AdGFP. GAPDH was used as a loading control. Representative images for three independent experiments are shown. (C) Nuclear extraction from Huh7 cells was subjected to Western blot to detect the expression of Notch3 protein after infection with AdC/EBPα or AdGFP. Representative images for three independent experiments are shown. (D) C/EBPα directly interacted with the first intron of Notch3 gene in vitro. Huh7 cells were treated with AdC/EBPα for 48h, and cell lysates were harvested and subjected to ChIP assay using the C/EBPα antibody. The purified DNA from ChIP assay was subjected to quantitative real-time PCR. Triplicate experiments were done, and the results were expressed as mean±SD. Notch3: neurogenic locus notch homolog protein 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ChIP: chromatin immunoprecipitation. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Fig. 5 Overexpression of C/EBPα inhibits Notch3/Hes1/p27 cascades. (A) Huh7 cells were treated with AdC/EBPα or AdGFP for 72h and cell lysates were analyzed by Western blot using anti-Hes1 or anti-p27 antibodies. GAPDH was used as an internal control for protein. Representative images for three independent experiments are shown. (B) Huh7 cells were transfected with pcDNA3.0-Hes1 or pcDNA3.0 for 6h as described in the experimental procedures, followed by AdC/EBPα or AdGFP infection and then seeded into 96-well plates. Cell numbers were assessed by Cell Counting Kit-8 analysis. The data shown are representative of three independent experiments. The P values were derived from statistical analyses made among the four groups, as determined by a one-way ANOVA. **P<0.01, NS, no significance. (C) Sections from formalin-fixed orthotopic liver tumour tissues from AdC/EBPα-treatment or AdGFP-treatment mice were stained by immunohistochemistry for Notch3, Hes1 and p27 (magnification 400×). Examples of strongly positive-stained cells are indicated by black arrows. Scale bars: 50μm. Hes1: hairy and enhancer of split 1; Notch3: neurogenic locus notch homolog protein 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S1 C/EBPα abolishes the tumourigenicity of Huh7 cells. (A) Huh7 cells expressing C/EBPα or GFP were injected subcutaneously into the right and left armpits of each nude mouse. The tumour nodules were collected at six weeks after subcutaneous implantation with Huh7 cells. (B) The HCC-free survival of mice was analyzed by the Kaplan–Meier method and compared using the long-rank test (n=7). *P<0.05 (compared to AdGFP). HCC: hepatocellular carcinoma. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S2 C/EBPα maintains the expression of liver-specific genes. (A) Real-time PCR analysis revealed that overexpression of a cluster of hepatocyte marker genes was provoked along with increased expression of exogenous C/EBPα in Huh7 cells versus the GFP-infected group 72h after adnenovirus-mediated C/EBPα gene transfer. *P<0.05, **P<0.01 (compared to AdGFP). (B) The expression of hepatocyte marker genes were detected by real-time PCR after knockdown of C/EBPα by siRNA in Huh7 cells for 72h. *P<0.05, **P<0.01 (compared to siNC). Three independent experiments were performed for A and B and the values represent mean±SD. siNC: siRNA negative control. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S3 Downregulation of C/EBPα and upregulation of Notch3 mRNA levels in human HCC. Real-time PCR analysis for C/EBPα and Notch3 mRNA expression was performed in 20 paired clinical HCC samples and their surrounding liver tissues. The mRNA expression levels were normalized against β-actin. Three independent experiments were performed and the data represent mean±SD. *P<0.05, **P<0.01 (compared to the adjacent non-tumour tissues). HCC: hepatocellular carcinoma; Notch3: neurogenic locus notch homolog protein 3. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S4 Over-expression of C/EBPα increased Numb mRNA expression. Numb mRNA expression levels were detected using normal PCR on Huh7 cells after infected with AdC/EBPα or AdGFP. At least three independent experiments were performed. Data are mean±SD. *P<0.05 (compared to AdGFP). Numb: numb homolog (Drosophila). Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S5 C/EBPα inhibited E2F target genes, accompanied with the induction of miR-122 and p21. Real-time PCR was used to detect the expression of cyclin D1, cyclinA2, calm2, p21 and miR-122 in Huh7 cells after infection with AdC/EBPα or AdGFP. E2F: E2F transcription factor; calm2: calmodulin 2; p21: cyclin-dependent kinase inhibitor 1A; miR-122: microRNA 122. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S6 C/EBPα inhibited the expression of Notch3 target genes. Real-time PCR was used to detect the expression of Hes1, c-Myc and Cyclin D1 in Huh7 cells after infection with AdC/EBPα or AdGFP. Data are mean±SD (n=3). *P<0.05, **P<0.01 (compared to AdGFP). Notch3: neurogenic locus notch homolog protein 3; Hes1: hairy and enhancer of split 1; c-Myc: v-myc myelocytomatosis viral oncogene homolog. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions

Supplementary Fig. S7 Knockdown of C/EBPα induced Notch3 and Hes1 expression. Knockdown of C/EBPα by siRNA increased the expression of Notch3 and Hes1 detected by real-time PCR. Three independent experiments were performed and data are mean±SD. *P<0.05, **P<0.01 (compared to siNC). Notch3: neurogenic locus notch homolog protein 3; Hes1: hairy and enhancer of split 1. Digestive and Liver Disease 2013 45, 844-851DOI: (10.1016/j.dld.2013.03.013) Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Terms and Conditions