BMC Cancer 2006, 6:109 http://www.biomedcentral.com/1471-2407/6/109 cFWaingecusetrereornu2sbloivterfotrisKsuI AesA0101 in HCC and counterpart non- Western blot for KIAA0101 in HCC and counterpart non- cancerous liver tissues. To investigate KIAA0101 protein expression level, 30 pairs of tumor and non-tumor liver tis- sues were analyzed. The KIAA0101 protein expression was lower in 25 out of 30 HCC tissues as compared with non- cancerous liver tissue (N: non-cancerous tissue; T: tumor tis- sue). Beta-actin was used an internal control. the intensity of band signal was generally proportional to the amount of loaded protein. Therefore, we concluded that our prepared rabbit polyclonal antibody for KIAA0101 was specific, and it could be used for further experiments. nFKoiIgAhuiAsrteo01c3h0e1meixsptrryes(sISioHn) ienxtaismsuineatairornay was analyzed by immu- KIAA 0101 expression in tissue array was analyzed by immu- nohistochemistry (ISH) examination. The tissue array showed different levels of KIAA0101 protein expression in paired HCC and non-cancerous tissues, liver cirrhosis and normal liver tissues by ISH method. Weak expression of KIAA0101 was observed in HCC; while strong expression in its counterpart non-cancerous liver tissue. In 8 out of 13 liver cirrhosis and all 10 normal liver tissues, KIAA0101 protein expression level were strong(+++) or moderate(++) level. KIAA0101 protein was down-regulated in HCC To demonstrate KIAA0101 expression pattern at the pro- tein level in human HCC, 30 human HCC (T) and matched noncancerous liver tissues (NT) from the same patients were analyzed by Western blot (Figure 2). The protein band intensity was compared after being normal- ized with that of the beta-actin internal control. It was striking that the protein expression level of KIAA0101 in HCC tissues was reduced as compared with that of non- cancerous tissues among 25 out of 30 paired samples. We have conducted the tissue array by immunohistochemical survey(ISH) for further validation. Fig 3 illustrated the dif- ferent intensity between HCC and matched non-cancer- ous tissues, liver cirrhosis and normal liver tissues by ISH method. As demonstrated in table 1 (see Additional file 1), the tissue array showed the signal intensity was weak(+) or negative(-) in 107 out of 161 HCC tissues; while the weak (+) or negative (-) signals were seen in only 24 out of 161 samples of non-cancerous liver tissues. Therefore, the KIAA0101 protein expression was much lower in the HCC tissues as compared with the non-can- cerous tissues (p < 0.01). In addition, in 8 out of 13 of liver cirrhosis tissues and all 10 normal liver tissues, KIAA0101 protein was expressed varying from moder- ate(++) to high(+++) level. However, the KIAA0101 pro- tein level in HCC tissues was obviously reduced. Furthermore, we have attempted to investigate if the down-regulation of KIAA0101 protein expression had any correlation with the patients' background information, such as age, sex, histopathological grading, tumor size and HBV infection. However, we have not found any signifi- cant association between the clinical or pathological state of HCC with the KIAA0101 expression (see Additional file 2). Localization of KIAA0101 in mitochondria The bioinformatics has implicated that KIAA0101 was possibly localized in mitochondrial. To verify this predic- tion, we have performed immuno-fluorescence survey on SMMC7721 cells by using the polyclonal rabbit antibody. KIAA0101 immuno-fluorescence was exhibited in a speckled pattern in the cytoplasm. The distribution of KIAA0101 immuno-fluorescence appeared to be similar to the distribution of mitochondria. In dual-labeling experiments, KIAA0101 immuno-fluorescence partially coincided with the image generated by a labeled mono- clonal antibody that recognized cytochrome oxidase sub- unit I, an inner mitochondrial membrane protein. The partially co-localization of KIAA0101 with mitochondrial proteins suggested that KIAA0101 was localized predom- inantly in mitochondria (Fig 4). Meanwhile, we used the specific mouse anti-mitochondrial (Neomarkers) to enrich the mitochondrial proteins from total cell proteins preparation. After precipitated by the protein A/G beads, the enriched mitochondrial proteins were subjected to Page 4 of 8 (page number not for citation purposes)