BMC Cancer 2006, 6:109 http://www.biomedcentral.com/1471-2407/6/109 LFoigcualrizeat4ion of KIAA0101 predominantly in mitochondria Localization of KIAA0101 predominantly in mitochondria. HCC SMMC 7721 Cells were seeded on slip for dual immun- ofluorescence analysis. Primary antibodies for cytochrome oxidase I and KIAA0101 were detected using FITC- and TRITC- conjugated secondary antibodies, respectively. Yel- low fluorescence in the overlay image indicated the co-locali- zation of the red fluorerscence of KIAA0101 with the green fluorescence of mitochonriion specific of cytochrome oxi- dase I. Western blot with KIAA0101 rabbit antibody. Fig 5 showed that there were strong signal in lanes loaded by enriched mitochondrial proteins; however, there were only very weak signal in the un-sorted total cell proteins. From these two different experiments, we could conclude that the KIAA0101 protein was predominantly located in the mitochondria of human HCC SMMC 7721 cells. Cell growth and cell cycle analysis For better understanding if the KIAA0101 has any effect on cell growth, we employed the pMACS KKII system to isolate the KIAA0101 over-expressed cells after transient KIAA0101 cDNA transfection into two different HCC cell line: Hep3B and HepG2. The isolated KIAA0101 expres- sion positive (+) cells and the KIAA0101 negative (-) con- trol cells were seeded into the 96-well plate. We measured the absorbance at the 450 nm with the CCK-8 kit each day. As illustrated in Fig 6, the KIAA0101 over-expression could inhibit the growth of both HCC cell line: HepG2 and Hep3B (P < 0.05). Meanwhile, as indicated in Fig 7A, the sorted KIAA0101 expression positive and negative cells were analyzed by FACS and the results demonstrated SeFwHWMniegrerpMiusecGtrChem2er7dinr5t7eopb2scrlp1oheotacenftoidinvrreHfiKralyaelI;cApe3tnGAiora20inc1rdha0een14sddppweptrcroeootrttiaetveeleitinclnoyesi)tlnalflrpmocreimotlotleScpiMhnrosMtn(eCd1irnai7anf7rld-2o1m2and Western blot for KIAA0101 protein in mitochondrial- enriched protein fraction and total cell proteins (1 and 2 were mitochondrial enriched proteins from SMMC 7721 and HepG2 respectively; 3 and 4 were total cell protein from SMMC 7721 and HepG2 respectively). TFHriCgaunCsriceen6ltl gerxopwretshsionnviortfoK(IApA<00101 protein inhibited the Transient expression of KIAA0101 protein inhibited the HCC cell growth in virto (p < 0.05). pcDNA3.1/A+0101 and pMACSKKII plasmids were co-transfected into the HepG2 and Hep3B cells. Meanwhile, the pcDNA3.1/A and pMACSK- KII plasmids were co-transfected and isolated as control. After about 48 hours, the KIAA0101 expression positive cells were selected by magnetic microbeads through column and seeded into 96-well plates. At every 24 hours, the cells' growth absorbance was measured with CCK-8 kit. that the cell population at S phase of KIAA0101 positive cells was reduced by nearly 8% as compared with the neg- ative cells and the difference had statistical significance(P < 0.05). The percentage of G0/G1 phase in KIAA0101 pos- itive (+) cells is 64.38% ± 1.46, while in negative cells the proportion of phase G0/G1 is 54.7% ± 1.43. The differ- ence was of statistical significance (p < 0.05, Fig 7B). The percentage of G2/M phase in KIAA0101 positive (+) cells is 14.69% ± 0.62 while in negative cells the proportion is 15.21% ± 0.51 with no statistical difference (Fig 7C). Taken together, the FACS result was well consistent with the retardation of HCC cell growth after over-expression of KIAA0101 protein. Discussion KIAA0101 has been recently reported as a novel gene related to thyroid [7], non-small cell lung carcinoma[8] and colon cancer[9,10]. In these reports, the alteration of expression at mRNA level was documented, either up-reg- ulated [7-9] or down-regulated [10] in cancers described. Page 5 of 8 (page number not for citation purposes)