Wen-Li et al. Journal of Experimental & Clinical Cancer Research 2012, 31:92 http://www.jeccr.com/content/31/1/92 Page 3 of 9 F1F0 ATPase β subunit was named as McAb7E10 and used in further experiments. Western blotting and BIAcore analysis Cellular proteins were extracted in 40 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100 and supplemented with a cocktail of protease inhibitors. Equal amounts of protein were resolved on 12% SDS-PAGE gels then transferred to a PVDF mem- brane. After blocking with 5% non-fat milk, the mem- branes were incubated with McAb7E10 antibody overnight at 4°C, then with HRP-conjugated sheep anti- mouse IgG secondary antibody (Vector, Burlingame, CA, USA). After washing, the protein bands were visualized using the SuperEnhanced chemiluminescence detection kit (Applygen Technologies Inc., Beijing, China) and X- ray film (Kodak,NY,USA). The binding and dissociation kinetics of McAb7E10 with the recombinant ATPase β subunit were determined using a BIAcore surface plasmon resonance instrument (Pharmacia, Uppsala, Sweden) [27-31]. Briefly, 1400 RU of the recombinant ATPase β subunit (25 ug/mL in 10 mmol/L sodium acet- ate, pH 4.5) were covalently bound through amino groups to a CM5 sensor chip [32-34]. ATPase activity assay 1*104 cells per well were equilibrated with serum-free medium at 37°C with 5% CO2 overnight, respectively, in 96-well plates. Then the cells were treated with different concentrations of McAb7E10, oligomycin (Sigma, St. Louis, MO, USA), a known inhibitor of ATPase F1 or mouse IgG for 30 min. The cells were then incubated with adenosine diphosphate (Sigma, St. Louis, MO, USA) for 60 s, and supernatants were removed and assayed for ATP production using a bioluminescence assay kit (Invitrogen, Carlsbad, CA, USA). Samples were injected with the ATP assay mixture (Promega, Madison, WI, USA) and incubated for 10 min to stabilize the lu- minescence signal. Recordings were made in an Analyst HT (Molecular Devices, Sunnyvale, CA, USA) over a 20 s period. Data are expressed as moles of ATP per well based on standards determined under the same condi- tions during each experiment. Cell proliferation assay Acute myeloid leukemia (AML) cells (MV4-11 and HL- 60) were seeded in 96-well plates at 50,000 cells per well and 5–50 ug/mL mouse control IgG or 5–50 ug/mL McAb7E10 antibody was added. After 24, 48, 72, 96 or 120 h, 20 μL 5 mg/ml MTT (3-(4,5-dimethylthiazol-2- yl)-2,5- diphenyltetrazolium bromide) solution was added to each well, incubated at 37°C for 4 h, then the media was removed and 200 μL dimethylsulfoxide (DMSO) was added. Optical density (OD) values were measured at 490 nm using a scanning multi-well spec- trophotometer (BioRad Model 550, Hercules, CA, USA), and the survival rates of McAb7E10 treated cells were calculated relative to the control antibody treated cells. All experiments were performed in triplicate and repeated twice. The results were analyzed using ANOVA and the Student-Newman-Keuls tests, p < 0.05 were con- sidered significant. Cell cycle analysis Cells were harvested and a single cell suspension was prepared in buffer (PBS + 2% FBS), washed twice and adjusted to 1 × 106 cells/ml. Aliquots of 1 ml cell sus- pension were placed in 15 ml polypropylene V- bottomed tubes and 3 ml cold absolute ethanol was added to fix the cells for at least 1 h at 4°C. Cells were washed twice in PBS, 1 ml propidium iodide staining solution was added to the cell pellet, mixed well, and 50 μl RNAse A stock solution was added and incubated for 3 h at 4°C before flow cytometry analysis was performed. Cell apoptosis analysis Cell apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Cat. No 556570; BD Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, incubated with mouse IgG or McAb7E10 antibody for 48 hours, then cells were washed twice with cold PBS, resuspended in 1x Binding Buffer at 1 × 106 cells/ml and a 100 μl (1 × 105 cells) ali- quot was transferred to a 5 ml culture tube. 5 μl Annexin V and 10 μl vital dye was added, gently mixed, incubated for 15 min at RT in the dark, then 400 μl of 1x Binding Buffer was added to each tube and immedi- ately analyzed by flow cytometry. All experiments were performed three times. Statistical analysis All data are presented as mean ± SD. Statistical analysis was performed using SPSS statistical software (SPSS Inc, Chicago, IL, USA), p ≤ 0.05 were considered significant. Results and discussion The ecto-ATPase β subunit is expressed in cell lines from hematologic malignancies The ATP synthase β subunit is known to be constitu- tively expressed in the inner mitochondrial membrane of normal cells, and ectopically expressed in primary cultured endothelial cells [3-7]. Liver carcinoma cells and lung carcinoma cells also express the ATP syn- thase β subunit on their cell surface [18,21]. In this study, we found that the ATP synthase β subunit is upregulated and ectopically expressed on the cell sur- face of human AML cells. Using flow cytometry, the β