1. Epigenetic Inhibition of Nuclear Receptor Small Heterodimer Partner Is Associated With and Regulates Hepatocellular Carcinoma Growth 
Nan He, Kyungtae Park, Yuxia Zhang, Jiansheng Huang, Shan Lu, Li Wang 
Gastroenterology 
Volume 134, Issue 3, Pages (March 2008)
DOI: /j.gastro
Copyright © 2008 AGA Institute Terms and Conditions

2. Figure 1
SHP promoter hypermethylation in HCC. (A) Left, Northern blot of 10 matched pairs of normal surrounding tissues (s) and HCC specimens (h) probed with hSHP cDNA. Right, immunohistochemistry analysis of hSHP protein in HCC. Blue, DAPI; red, SHP staining. (B) SHP promoter hypermethylation profiling in HCC. The top diagram shows the SHP gene and the location of nested primers used for bisulfite genomic sequencing. Exon 1 is shown as a dashed line, the transcription start site is shown as an arrow, and CpG sites are shown as tick marks. F1/R1 are used to amplify the promoter region containing 6 orphaned CpGs, whereas the F2/R2 are used to amplify the entire region of exon 1. All 20 CpG sites in the predicted CpG island region of the SHP gene were analyzed by bisulfite genomic sequencing for their methylation status. White and black circles denote unmethylated and methylated CpG sites, respectively. Four cloned PCR products were sequenced to determine the percentage of methylation of the CpG sites in the regions analyzed. (C) Summary of 5-methycytosine levels for each CpG site obtained by bisulfite genomic sequencing of the SHP gene in 10 matched pairs of normal and HCC specimens.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
Correlation of methylation in the promoter region with silencing of the SHP gene in HCC cells. (A) Methylation status of the 6 CpGs of the SHP gene promoter in HCC cell lines as determined by bisulfite sequencing. Four different clones from each line are presented and methylated CpG sites are represented by black circles. (B) Northern blot analysis of SHP mRNA after treatment of HepG2 cells with demethylating agent Aza or histone deacetylase inhibitor TSA. (C) The hSHP reporter (hSHPp Luc) was methylated in vitro with 1 U of SssI methylase, transfected with the human LRH-1 (100 ng) expression vector in Aza (2 μmol/L) untreated or treated cells. Luciferase activity was determined and normalized by β-gal activity. For the Aza group, cells were treated with Aza 1 day before the transfection, and were fed fresh medium containing Aza every day. ▫, -LRH-1; ▪, +LRH-1. (D) ChIP assays. The hSHPp Luc was methylated by SssI, and Huh7 cells were transfected with the hSHPp Luc (nonmethylated or methylated) in the absence or presence of Aza. n.s., nonspecific primers located 4 k upstream of the SHP promoter. Input serves as control. Left, positions of primers used for ChIP analysis. Gray bars indicate 3 LRH-1 binding sites within the 6 CpG sites. (E) ChIP analysis of enrichment of MeCP2, MBD1, and sin3A on SHP chromatin by SssI treatment. The results are expressed as the percentage of immunoprecipitate (ip) over total input DNA used. ▫, Con; ▪, SssI.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
SHP inhibition of tumor formation. (A) HepG2 cells were infected with either GFP-control virus (GFP-Ad) or SHP-adenovirus (SHP-Ad), and foci formation was quantitated in soft agar. Green fluorescence was used to identify GFP-Ad– or SHP-Ad–infected cells. Green arrows in the SHP-Ad group represented foci formed from cells not infected with SHP-Ad (no green fluorescence detected from these cells). Only foci formed GFP- (▫) or SHP (▪)-infected cells were counted and used for statistical analysis (right panel). (B) Huh7 cells (2 × 106) were infected with either GFP-Ad (top) or SHP-Ad (bottom), grafted subcutaneously in the dorsa of athymic mice, and tumor growth was monitored at day 10 as indicated (left panel). A second group of mice received an intratumoral injection of 109 pfu of GFP-Ad or SHP-Ad at 10 days after tumor implantation and were analyzed at day 25. Tumor volume (right panel) was monitored. ○, GFP; ●, SHP. (C) Huh7 cells were infected with either GFP-Ad or SHP-Ad, then treated with TNFα or AHPN, or starved for 72 hours. The number of apoptotic cells was quantified by Annexin V staining. EtOH and dimethyl sulfoxide (dmso) are solvents for cycloheximide and AHPN, respectively. ▫, GFP; ▪, SHP.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

5. Figure 4
Real-time PCR analysis of the SHP gene expression in HCC. (A) Total RNA was isolated from a total of 19 pairs of normal surrounding tissue (▫, surr. tiss) and the corresponding HCC (▪) and SHP mRNA levels were analyzed by real-time PCR. (B) Real-time PCR results of the mRNA levels of SHP in 19 normal surrounding liver tissues compared with that of 57 HCC pathologic specimens. (C) The expression pattern of SHP in HCC specimens was compared between disease groups including hepatitis C (HepC) or hepatitis B (HepB). “Other” represents patients with cryptogenic cirrhosis, hemochromatosis, or other disorders leading to an increased risk for HCC.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions