Table 1.
Primary antibodies.
Table 2.
Result of cDNA microarray analysis of EpCAM+ cells between normal and DDC-fed mouse livers.
Fig 1.
Expression profiles of SAMD5 in mouse.
(A). Real-time RT-PCR analysis of Samd5 mRNA in EpCAM+ cells isolated from normal and DDC-fed mouse livers. (B) Real-time RT-PCR analysis of Samd5 mRNA in normal liver (n = 3), 70% PHx liver (n = 4), chronically injured liver by CCl4 (n = 5) or DDC diet (n = 4). The upregulation of SAMD5 expression is outstanding in DDC-fed mouse liver. Data are means ± standard error. *P <0.05; **P <0.01. (C). Immunostaining of SAMD5 and CK19 for DDC-fed mouse liver. SAMD5 is markedly expressed in a part of large bile ducts and PBGs at the hepatic hilum (arrowheads), whereas it is not detected in numerous ductular cells located in parenchymal region (arrows). Bars = 50 μm.
Fig 2.
IHC and PAS staining for serial sections of DDC-fed mice liver.
(A) Immunostaining of SAMD5 and CK19 for DDC-fed mice liver. SAMD5 is markedly expressed in several PBGs (solid arrows) and columnar mucus-producing cholangiocytes (open arrows) at the hepatic hilum, whereas SAMD5 is not detected in cuboidal ductular cells (arrow heads). (B) PAS staining for the serial section of panel (A). Mucin is stained violet, while deposition of Iron and bile plug is observed as red and black agglutination. Mucin is detected in hilar large bile duct and PBG, but not in cuboidal ductular cells (arrow heads). The lower panel is a magnified image of the upper panel. Bars = 100 μm.
Fig 3.
Expression profiles of SAMD5 and EpCAM in extrahepatic bile duct.
(A) Double immunostaining of the vertical section of normal common bile duct with anti-SAMD5 and anti-EpCAM antibodies. SAMD5 is clearly detected in PBGs (arrows), but not in the luminal epithelium of normal common bile duct. (B, C) Double immunostaining of the vertical (B) and horizontal (C) sections of DDC-fed common bile duct with anti-SAMD5 and anti-EpCAM antibodies. SAMD5 is highly expressed in both PBGs (arrows) and the luminal epithelium of dilated common bile duct (arrowheads).
Fig 4.
Immunostaining of human liver tissue sections with anti-SAMD5 antibody.
SAMD5 was stained for the paraffin-embedded sections of normal large bile duct at the hepatic hilum (A), intrahepatic CC (B), hilar CC (C and D), and extrahepatic CC (E). (A) Low or medial cytoplasmic staining of SAMD5 is observed in normal hilar large bile duct (asterisk). (Original magnification X200) (B) While the intrahepatic cholangiocytes show the cytoplasic staining of SAMD5 (arrow), the poorly-differentiated ICC exhibits striking nuclear staining of SAMD5. (Original magnification X100) (C) SAMD5 is stained in the nuclear of both poorly-differentiated ICC (p-ICC) and well-differentiated ICC (w-ICC) at the hepatic hilum. (Original magnification X200) (D) The cancerous cells invading hilar large bile duct show nuclear staining of SAMD5 (arrowhead). (Original magnification X200) (E) The papillary and moderately-differentiated tubular adenocarcinomas in the common bile duct exhibit nuclear staining of SAMD5. (Original magnification X200).
Fig 5.
Expression profiles of SAMD5 in human HCC and CC cell lines.
(A) the relative expression of SAMD5 gene in four CC cell lines (HuH28, TFK1, RBE and TKKK) and one HCC cell line (HuH7) to normal biliary epithelial cell (BEC) by quantitative RT-PCR. SAMD5 mRNA was increased in all CC cell lines, but not expressed in HuH7. (n = 3; *P <0.05, compared to BEC) Data are mean ± standard error. n.d.: not detected (B). Immunocytochemical images of SAMD5 for CC cell lines. SAMD5 is visualized and localized at the nuclei of TKKK and RBE. Bars = 50 μm. (C and D) Images of exogenously introduced FLAG-tagged SAMD5 in HuH7 (C) and HuH28 (D) by Immunocytochemical staining. Overexpressed SAMD5 translocated to the nuclei of each cell. Bars = 50 μm.
Fig 6.
Relationship between SAMD5 expression and cell cycle in CC cell line.
(A) Real-time RT-PCR analysis of SAMD5 mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of SAMD5 in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of SAMD5 mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *P <0.05; **P <0.01; ***P <0.001.