the internal standard (ISTD) was monitored at m/z 299.3. Calibration solution of neat OA was made in 1/1 v/v water:MeOH at 0.01, 0.1, 1, 5, 10, 50, and 100 mg/ml levels containing 0.5 mg/ml internal standard. At the 100 mg/ml calibration level, the peak saturation occurs. 16.67 ml of original protein-OA solution was diluted 60 times to give 1 ml of sample solution that also contained 0.5 mg/ml of ISTD. For quantitative analysis, 25 ml of calibrator or protein-OA sample solution was injected into a LC-MS system comprised of a capillary LC column (Supelco C18 column (50 mm60.5 mm65 mm), Sigma) coupled via electrospray to a triple quadrupole mass spectrometer utilizing single ion monitoring technique (SIM) in negative ionization mode, monitoring OA at m/z 281.2, and the internal standard (ISTD) at m/z 299.3. The protein-OA sample was run in triplicate. A linear calibration curve with a weighting index of 1/X was used, and an R2 of 0.9999 was observed. Cell culture Human lung adenocarcinoma cells A549 were cultured in RPMI-1640 medium (HyClone, Thermo Fisher, Logan, UT) supplemented with 2 mM glutamine, 100 IU/ml penicillin- 100 mg /ml streptomycin (HyClone), 50 mg/ml gentamicin (Gibco/Invitrogen, Grand Island, NY), 1% non-essential amino acids (HyClone), and 10% (v/v) fetal bovine serum (FBS, Gibco). SK-BR-3 and MDA-MB468 cells were cultured in McCoy’s 5A media (Gibco) supplemented with 10% FBS and antibiotics. MCF- 7 cells were grown in RPMI 1640 (Gibco) and supplemented with 10% FBS and antibiotics. MCF-10A immortalized, non-transformed mammary epithelial cells (obtained from Dr. Esta Sterneck’s laboratory, NCI- Frederick) were cultured in DMEM/F-12 1:1 (Gibco) supple- mented with 100 IU/ml penicillin- 100 mg/ml streptomycin (HyClone), FBS, 10 mg/ml insulin (Sigma), 100 ng/ml Cholera toxin (Calbiochem, La Jolla, CA), 0.5 mg/ml Hydrocortisone (Sigma), 20 ng/ml EGF (Invitrogen) and 1 mM CaCl2. Cell death assay Cell viability was determined using the Trypan blue exclusion method. Cells, 5x104 cells per well, were plated in a 48-well plate (Corning Incorporated, Corning NY). After 24 h, media was removed and replaced with fresh media without FBS, and the different protein/ protein complexes were added and incubated for the indicated lengths of time. Cells were incubated with 30 mM molten globule protein alone or complexed with OA for 3 h (the first hour of incubation was without FBS). After treatment, floating cells from conditioned media and trypsinized cells were pooled and pelleted. Cells were resuspended in 50 mL of PBS, and the cell suspension was mixed with an equal volume of 0.4% Trypan blue stain (Gibco). Clear, viable cells were counted microscopically in Kova glasstic slides with grid chamber References 1. Brodbeck U, Denton WL, Tanahashi N, Ebner KE (1967) The isolation and identification of the B protein of lactose synthetase as alpha-lactalbumin. J Biol Chem 242: 1391–1397. 2. Qasba PK, Safaya SK (1984) Similarity of the nucleotide sequences of rat alpha-lactalbumin and chicken lysozyme genes. Nature 308: 377– 380. 3. Dolgikh DA, Gilmanshin RI, Brazhnikov EV, Bychkova VE, Semisotnov GV, et al. (1981) Alpha-Lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett 136: 311–315. 4. Kuwajima K (1996) The molten globule state of a-lactalbumin. FASEB J 10: 102–109. 5. Ramakrishnan B, Qasba PK (2001) Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the b1-4-galactosyl- transferase-I. J Mol Biol 310: 205–218. 6. Ramakrishnan B, Boeggeman E, Ramasamy V, Qasba PK (2004) Structure and catalytic cycle of beta-1,4-galactosyltransferase. Current Opinion in Structural Biology 14: 593–600. Studies with Site-Specific Labeled HAMLET (Hycor, Garden Grove, CA). Cell number is expressed as a percentage of the untreated control cells. For Annexin V detection, cells were incubated with 30 mM molten globule protein alone or complexed with OA. After treatment, floating and trypsinized cells were pelleted and washed once with binding buffer and stained with Annexin V-FITC (ApoAlert, Clontech, Mountain View, CA) according to the manufacturer’s instructions. Cells were quantified in a Becton Dickinson FACScalibur cytometer and CellQuest software. Confocal fluorescence microscopy Cells were plated on an 18-well slide (ibiTreat, Ibidi Integrated BioDiagnostics, Munich, Germany) and grown for 2 nights (2000 cells/well). Media was replaced with mg-hLA complexed with OA (20 mM unlabelled or 10 mM labeled); or mg-hLA alone (20 mM unlabelled or 10 mM labeled) and cells were then incubated at 37uC in media without serum. Same procedure was followed for aD-hLA. For anti-fibrillarin staining, cells were incubated for 4 h, and then slides were fixed for 15 min with 4% paraformaldehyde in PBS. After rinsing with PBS, cells were permeabilized in a PBS solution containing 0.1% TritonX-100, 2 mg/ml BSA, and 1 mM sodium azide for 5 min, and then blocked for 1 h in a PBS blocking solution (0.05% Tween 20, 2 mg/ml BSA, 1 mM sodium azide). Samples were incubated overnight with anti-fibrillarin mouse monoclonal antibody (Invitrogen), 1:500, in blocking solution at room temperature in a humidified chamber. After 3610-min wash, the sample was incubated with 1:1000 anti- mouse-Alexa Fluor 555 antibody (Invitrogen) in blocking solution. Following the washes, nuclei were stained with Hoechst 5 mg/ml for 15 min, and slides were mounted with mounting media (Ibidi). Confocal microscopy studies were performed with an Olympus Fluoview 1000 inverted microscope with lX81. Olympus FV100 2.0c and FV-ASW 1.6 viewer software were used for acquisition and analysis, respectively. Acknowledgments We thank Dr. Yuko Tsutsui for critical reading of the manuscript. We thank Mr. Sergey Tarasov and Ms. Marzena Dyba for CD support; Dr. Esta Sterneck for MCF-10A cell line, Dr. Ji Ming Wang for glioblastoma cell line, Ms. Kimberly Peifley and Dr. Stephen Lockett for confocal microscopy support; Mr. Timothy Waybright for MS; Dr. Athar Masood and Dr. Timothy Veenstra for LC-MS. Author Contributions Conceived and designed the experiments: PQ BR NM. Performed the experiments: NM BR EB. Analyzed the data: NM BR PQ. Contributed reagents/materials/analysis tools: BR EB PQ. Wrote the paper: NM BR PQ. 7. Qasba PK, Ramakrishnan B, Boeggeman E (2008) Structure and function of beta-1,4-galactosyltransferase. Curr Drug Targets 9: 292–309. 8. Lee AK, Rosen PP, DeLellis RA, Saigo PE, Gangi MD, et al. (1985) Tumor marker expression in breast carcinomas and relationship to prognosis: an immunohistochemical study. Am J Clin Pathol 84: 687–696. 9. Wrba F, Reiner A, Markis-Ritzinger E, Holzner JH, Reiner G, et al. (1988) Prognostic significance of immunohistochemical parameters in breast carcino- mas. Path Res Pract 183: 277–283. 10. Cohen C, Sharkey FE, Shulman G, Uthman EO, Budgeon LR (1987) Tumor- associated antigens in breast carcinomas: prognostic significance. Cancer 60: 1294–1298. 11. Thompson MP, Farrell HM, Mohanam S, Liu S, Kidwell WR, et al. (1992) Identification of human milk a-lactalbumin as a cell growth inhibitor. Protoplasma 167: 134–144. 12. Sharp JA, Lefe`vre C, Nicholas KR (2008) Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution. BMC Biol 6: 48–63. PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26093