BMC Cancer 2006, 6:109 http://www.biomedcentral.com/1471-2407/6/109 occurred in several cancers including thyroid [7], non- small cell lung cancer [8], and colon cancer [9]. This gene was possibly related to some mechanisms regulating cell proliferation and apoptosis [7,9]. Since the alteration of KIAA0101 expression reported so far was based on mRNA transcription, we studied the KIAA0101 protein expression level in human HCC as compared with the matched non-cancerous liver tissues by using an antibody prepared in our laboratory, and fur- ther investigated its subcellular localization in HCC cells and its biological effect on HCC growth and cell cycle. We found that KIAA0101 was remarkably down-regulated at protein level in HCC, and it was capable of inhibiting cell growth and blocking the transition from G1 to S phase in cell cycle. Methods Tissue samples and tissue array The human liver cancer samples and matched adjacent liver tissues were collected from the First Affiliated Hospi- tal of Zhejiang University (Hangzhou, PR China). The HCC cell lines were provided by our lab and cultured in standard conditions (10% fetal bovine serum, 5% CO2). Tissue array was prepared by our lab including 161 pairs of liver cancerous tissues and adjacent non-cancerous tis- sues, 13 liver cirrhosis tissues and 10 normal livers. All samples of collection were under consensus agreements, and were approved by the Ethical Review Committee of the World Health Organization Collaborating Center for research in Human Production. Antibody preparation KIAA0101 coding sequence was subcloned into the pEGFP plasmid and pET-32a prokaryotic expression vec- tor (His tag) separately and sequenced for confirmation. The primers for pEGFP vector are 5-ggagatctaacatggtgcg- gactaaag-3 and 5-gggtcgaccattctttttcatcatttg-3; the primers for pET-32a are 5-ggggatccgcaacatggtg cggactaaag-3 and 5- ggctcgagttctttttcatcatttg-3. The sequence underlined is restriction site for subcloning. Then the pET-32a+0101 recombinant expression plasmid was transformed into competent BL21 cells. Following induction of BL21 cells with 0.4 mM IPTG at 37°C for 4 hours, then the whole cells were lysised by the suspersonic equipment. The supernatant including the cell whole proteins were flowed through the Ni-coated column and the His-KIAA0101 fusion proteins were bound. After three times wash of the column, the His-KIAA0101 fusion protein was obtained by the elution buffer and was used for rabbit polyclonal antibody production according to the standard procedure. Western blotting The liver tumor and non-tumor samples were lysised in T- PER tissue protein extraction reagent (PIERCE) containing proteinase inhibitor cocktail (Roche) in 4°C for 30 min- utes. The debris was discarded and the supernatant including whole proteins were quantified by BCA kit (PIERCE) and subjected to 15% SDS-PAGE (40 ug). Next the proteins were transferred onto a nitrocellulose mem- brane (Schleicher & Schuell BioScience) and this mem- brane was blocked by the 5% milk dissolved in PBST for an hour. After incubation with the fist antibody and sec- ond antibody sequencely, detection was performed by using an ECL system (PIERCE). The rabbit anti-KIAA0101 antibody was used (1:1000) and GFP antibody (1:1000). Anti-β-actin antibody (1:10000, Sigma) was used as inter- nal control. Immuno-histochemistry The tissue array plate was blocked with 3% BSA 37°C for 1 hour, then probed with KIAA0101 antibody (1:100) at 4°C in a humidified chamber for 12 hours, followed rinsed three times by PBS and incubated with secondary rabbit antibody for 1 hour at 37°C (1:1000). This anti- body complex was visualized by 3, 30-diaminobenzidine (Zymed Laboratories Inc.). Sections were counterstained with hematoxylin for 5 min and mounted with Eukit (Cal- ibrated Instrument). The result was observed under a light microscope, and a semi-quantitative score for intensity was given to the different samples: +++, ++, + and -. Dual immuno-fluorescence and enriched mitochondrial protein Western blot Cultured HCC cells were seeded on the cover slips and incubated for 12 hours. Then the cells were probated with IgG-purified anti-KIAA0101 primary antibody (1:100) for half an hour in 37°C, after three times wash by PBS, the cells were incubated with TRITC-conjugated goat anti-rab- bit secondary antibody for an hour. Also the same cover slip were washed and antibody angnist cytochrome oxi- dase I was added at 1 mg/ml in block buffer, followed by FITC-conjugated goat anti-mouse IgG at 15 mg/ml in block buffer. The cells were viewed by epi-fluorescence with Axioskop 2 universal microscope. Digital images were captured using a CCD camera and analysized by ISIS system. For enriched mitochondrial protein Western blot, cells were rinsed twice with cold PBS and then lysed in radio- immune precipitation (RIPA) buffer for 15 min at 4°C. Cell lysates were clarified by centrifugation at 10,000 rpm for 10 min, and the supernatant was incubated with 1 ug anti-mitochondrial antibody (Neomarks) for overnight in 4°C. Then the immune complexes were precipitated by protein A/G-Sepharose beads and washed four times with Page 2 of 8 (page number not for citation purposes)