Volume 128, Issue 5, Pages 1369-1380 (May 2005) Human Homologue of Maid Is a Useful Marker Protein in Hepatocarcinogenesis  Taro Takami, Shuji Terai, Yuichiro Yokoyama, Haruko Tanimoto, Kunihiko Tajima, Koichi Uchida, Takahiro Yamasaki, Isao Sakaida, Hiroshi Nishina, Snorri S. Thorgeirsson, Kiwamu Okita  Gastroenterology  Volume 128, Issue 5, Pages 1369-1380 (May 2005) DOI: 10.1053/j.gastro.2005.03.014 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 HHM expression in experimental animal model of hepatocarcinogenesis. The rat CDAA diet model was used as an animal model of hepatocarcinogenesis to assess the expression of HHM by in situ hybridization (ISH) (A–C) and immunohistochemical analysis (D and E). (A) ISH of HHM mRNA for rat liver in the CDAA diet model (original magnification, ×40). Arrows indicate foci region, and arrowheads indicate HCC region. (B) Foci region (original magnification, ×100). (C) HCC region (original magnification, ×100). High levels of HHM mRNA expression were seen in foci (B) and HCCs (C). (D) Immunohistochemical localization of HHM in foci (arrows) (original magnification, ×100). (E) Immunohistochemical localization of GST-P protein in the serial section. Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Immunohistochemical localization of HHM in human AH, HCC, and cirrhotic liver tissue. Representative histology of AH (A), well-differentiated HCC (C), and cirrhotic liver tissue (E) (H&E stain; original magnification, ×40). B, D, and F show the immunohistochemical localization of HHM in serial sections. Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Immunohistochemical localization of HHM in human AH, HCC, and cirrhotic liver tissue. Representative histology of AH (A), well-differentiated HCC (C), and cirrhotic liver tissue (E) (H&E stain; original magnification, ×40). B, D, and F show the immunohistochemical localization of HHM in serial sections. Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effects of HHM on the cell cycle by flow cytometric analysis. Cells of each cell line (NHDF cells and HepG2 cells) were infected with Adeno-CMV-HHM or Adeno-CMV-LacZ at an MOI of 150 pfu/cell. The effects of HHM on cell cycle were determined by flow cytometric analysis. (A) Representative histogram of relative DNA content in NHDF cells infected with Adeno-CMV-HHM. Of the NHDF cells, 62.4% ± 0.9% were in the G0/G1 phase with 2C DNA content, 20.0% ± 1.0% in the S phase with DNA content between 2C and 4C, and 17.5% ± 0.7% in the G2/M phase with 4C DNA content. (B) Representative histogram of relative DNA content in NHDF cells infected with Adeno-CMV-LacZ (control). In NHDF cells infected with Adeno-CMV-HHM, the cell number in the G0/G1 phase was significantly (*P = .00014, n = 5) decreased, and that in the S phase was significantly (**P = .00029) increased when compared with controls. The cell number in the G2/M phase was not significantly different (P = .052). (C) HepG2 cells infected with Adeno-CMV-HHM. (D) HepG2 cells infected with Adeno-CMV-LacZ (control). In HepG2 cells infected with Adeno-CMV-HHM, the cell number in the G0/G1 phase was also significantly (***P = .00076, n = 12) decreased, and that in the S phase was significantly (****P = .00063) increased. The cell number in the G2/M phase was not significantly different (P = .085). Results are mean ± SD. Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 HHM knockdown analysis and colony formation assay. (A) Expression of HHM protein in HepG2 cells transfected with pcPURU6βi-HHM, which express HHM interference RNA, and pcPURU6βi-STOP (control). (B) Colony formation assay. HepG2 cells transfected with pcPURU6βi-HHM or pcPURU6βi-STOP were seeded into 60-mm-diameter culture dishes at the same density. After 2 weeks, colonies were counted as reported in the Materials and Methods section. For each cell line, a representative field on stereo microscopic inspection is shown. (C) Colony number of HepG2 cells transfected with pcPURU6βi-HHM or pcPURU6βi-STOP in soft agar (97.4 ± 5.6 vs. 149.2 ± 17.9, respectively; P = .00026, n = 5). Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Interaction of HHM and Jab1 proteins in KMC-1 cells. Both HHM and Jab1 were expressed in nuclei of KMC-1 cells. A nuclear protein lysate of KMC-1 cells was used in immunoprecipitation (IP) and Western blotting. The positive control lane consisted only of the nuclear protein lysate. The negative control lane consisted of the nuclear protein lysate, control IgG, and protein A/G-Agarose but not the primary antibody (A: anti-HHM, B: anti-Jab1). The IP lane consisted of the nuclear protein lysate, primary antibody (A: anti-HHM, B: anti-Jab1), control IgG, and protein A/G-Agarose. Western blotting was performed using the antibodies listed on the left. The positions of bands for Jab1 (A) and HHM (B) are indicated by arrows. Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Colocalization of HHM and Jab1 protein by immunofluorescent staining and immunohistochemical localization of Jab1 in human AH and HCC tissue. The colocalization of HHM and Jab1 proteins in KMC-1 cells (A–D) and HepG2 cells (E–H) were analyzed by immunofluorescent staining. (A and E) Red Cy-3 signal indicates the intracellular localization of HHM protein. (B and F) Green FITC signal indicates the intracellular localization of Jab1 protein. (C and G) Each nucleus was stained by DAPI. (D and H) Double fluorescent staining using red Cy-3 and green FITC signals confirmed the coexpression (yellow) of intranuclear HHM and Jab1 proteins (arrows indicated). (I and J) The immunohistochemical localization of Jab1 in AH (I) and well-differentiated HCC (J) tissue. (Original magnification, ×100). Gastroenterology 2005 128, 1369-1380DOI: (10.1053/j.gastro.2005.03.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions