PMC

Induction of GADD45β expression after 5-Aza-dC treatment. Forty-eight hours after treatment by different doses of 5-Aza-dC, expression of GADD45β in HepG2 and CL-48 cells was analyzed by Northern blot. The experiments were performed in triplicates and are presented as the mean ± SD. The probe for Northern blot was generated as mentioned in Material and Methods. GAPDH was used as an internal control for RNA loading. ImageQuant version 5.0 was used for quantification.

Identification of GADD45β proximal promoter region. The diagram illustrates the location of putative binding sites for the putative transcription factors. Fragments deleting each binding site were cloned into the pGL3 luciferase reporter plasmid. Relative luciferase activity for each promoter fragment is shown. Putative transcriptional factor binding sites are shown as blank boxes. Experiments were performed in triplicates and the results are presented as the mean ± SD.

Analysis of GADD45β promoter methylation in DNA from tissues and HepG2 cells. A: MSP analyses of methylation in DNA from HCC tissues, nonneoplastic liver tissues, and HepG2 cells. Top: The MSP results of nine cell lines. Middle: The MSP results of eight HCC samples. Bottom: Nonneoplastic liver tissues. The M indicates the hypermethylated PCR products and the U indicates unmethylated PCR products. B and C: Nucleotide sequencing of the third NF-κB and the putative inhibition region after sodium bisulfite treatment. •, complete methylation; □, partial methylation, ○, unmethylated.

Suppression of TGF-β and colony formation by p53-dependent GADD45β expression. A: The p53 expression was examined by Western blot. B: GADD45β expressing fluorescence green staining confirmed the transfection in HepG2 and Hep3B cells (Hep3B cell image not shown). C: The flow cytometry assay examined HepG2 and Hep3B without and with transfection of GADD45β was examined. D: The TGF-β examined by ELISA was described in the Materials and Methods. E: The colony formation assay was described in Materials and Methods. The number of colonies scored in the presence of pIRES2-EGFP vector was designated 100%. All of the experiments were performed at least three times with SD.

Analysis of GADD45β expression in HCC and nonneoplastic liver tissue. A: Northern blot was probed with a 222-bp PCR product that included GADD45β exon 3. The RT-PCR product was generated based on the GADD45β sequence AF078077 in GenBank. GAPDH was used as an internal control for RNA loading. RNAs were isolated from normal liver cell and HCC cells, eight HCC samples, and five matched nonneoplastic liver tissues. N, H, and Pt indicated nonneoplastic liver tissues, HCC tissues, and patient number, respectively. B: IHC study was used to confirm the diagnosis and expression level. Top: H&E-stained sample. The N indicates normal liver tissue and H indicates HCC tissue. Bottom: Stained for GADD45β and shows a diffuse yellowish tint, predominantly in cytoplasm of normal cells. The boundary between cancer tissue and noncancerous tissue was separated by fibrotic tissue. The bottom panel showed very low GADD45β expression in HCC tissue, compared with high GADD45β expression in normal liver tissue. Original magnifications, ×40.