Herceptin Conjugate Induces Tumor Cell Necrosis using amicon-50 KDa (Millipore, Billerica, MA, USA). SMCC- conjugated Herceptin was made as described in the manufactur- er’s SMCC instruction (Thermo Scientific, Rockford, IL, USA). tain View, CA, USA). After 15 min incubation in 37uC, cells were observed under a fluorescence microscope (Olympus American, Inc., Center Valley, PA, USA). SDS-PAGE analysis Volumes containing 40 mg of protein were mixed with the same amount of 26 SDS loading buffer in the presence and absence of 10% DTT reducing reagent and added to wells of a 6% polyacrylamide gel, followed by the application of an electric field. The gel was then incubated with Coomassie Brilliant Blue R250 followed by destaining solution (10% acetic acid and 20% methanol). Images were captured with Quantity One version 4.4.1 software (Bio-Rad Laboratories, Hercules, CA, USA). Purification of Herceptin trimer complex using HPLC The conjugation products were purified by a HPLC system using Waters Alliance 2690 with ultraviolet-absorbance detection at 280 nm. Trimers, dimers, and monomers were separated on the 30067.8 mm Bio-Sil SEC-400-5 size exclusion column (Bio-Rad, Richmond, CA, USA) and were equilibrated in phosphate buffer (0.05 M monosodium phosphate [NaH2PO4], 0.05 M disodium hydrogen phosphate [Na2HPO4], and 0.15 M sodium chloride [NaCl], pH 6.8) at a flow rate of 0.5 mL/min. The protein fractions obtained were dialyzed overnight in distilled water at 4uC and lyophilized. A standard protein molecular mass profile was established using gel filtration standards (Bio-Rad) ranging from 1.3 to 670 KDa to identify the monomer, dimer, and trimer antibody peaks. Western blot analysis One microgram of Herceptin and HPLC-separated Herceptin dimers or trimers from EDC- or SMCC-conjugates were mixed with 26 SDS-PAGE sample loading buffer, added to a 6% polyacrylamide gel, subjected to SDS-PAGE, and then transferred to a TransBlot Transfer Medium nitrocellulose membrane (Bio- Rad Laboratories). Immunoblotting of the membrane was performed with a 1:100 dilution of polyclonal goat antihuman IgG containing secondary horseradish peroxidase. The peroxidase signal was generated by electrochemiluminescence using Western Lightning ECL (PerkinElmer, Inc., Walthman, MA, USA) and visualized with a Kodak Image Station 440CF, using the one- dimensional Image Analysis software version 3.6 (NEN Life Science Products, Inc., Boston, MA, USA). Live/dead cell assay For the live/dead cell staining, cells were washed three times after treatments. The cell-permeable green fluorescent dye for staining the live cells and PI for staining the dead cells were each diluted in the staining buffer according to manufacturer’s directions (Live/Dead Cell Staining Kit, BioVision, Inc., Moun- References 1. Press MF, Finn RS, Cameron D, Di Leo A, Geyer CE, et al. (2008) HER-2 gene amplification, HER-2 and epidermal growth factor receptor mRNA and protein expression, and lapatinib efficacy in women with metastatic breast cancer. Clin Cancer Res 14: 7861–7870. 2. Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, et al. (2002) Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 20: 3095–3105. 3. Goodwin PJ, Ligibel JA, Stambolic V (2009) Metformin in breast cancer: time for action. J Clin Oncol 27: 3271–3273. 4. O’Brien NA, Browne BC, Chow L, Wang Y, Ginther C, et al. (2010) Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib. Mol Cancer Ther 9: 1489–1502. MTT assay Cells (36104 per well, 5 replications for each treatment) with different treatments were incubated for 72 h. A quantity of 20 mL of MTT solution (5 mg/mL) was added to each well and incubated for 5 h prior to adding 200 mL of dimethyl sulfoxide (DMSO) to stop the reaction. The light absorbance was determined at 540 nm using a spectrometer (Packard Instrument, Inc., Downers Grove, IL, USA). BrdU flow assay Cells (36104 per well, 5 replications for each treatment) with different treatments were incubated for 72 h. BrdU flow assay (APC BrdU flow kit, BD Biosciences, San Jose, CA, USA) was performed according to the manufacturer’s directions. Caspase activity assays CaspGLOW Green Caspase Staining Kit (BioVision, Inc.) was used to detect activated caspase-3, -8, or -9. The stained cells were analyzed using FACSCalibur flow cytometer (BD Biosciences). A total of 10,000 cells per sample were acquired, and data samples were analyzed using FlowJo software (version 7.6.1). Cell necrosis and apoptosis assays via flow cytometry After treating the cells for 48 h, cells were gently trypsinized and washed once with serum-containing media. The cell pellets were washed with cold PBS (calcium [Ca2+] and magnesium [Mg2+] free) and binding buffer. The cell pellets were then resuspended in 500 mL binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazi- neethane sulfonic acid [HEPES], 140 mM NaCl, and 2.5 mM calcium chloride [CaCl2], pH 7.4) containing 5 mL FITC-annexin v and 5 mL PI in each sample, and cells were incubated on ice for 15 min. Samples were analyzed using flow cytometry. Statistical analysis A one-way analysis of variance (ANOVA) test was used to determine statistical significance, followed by a post-hoc Tukey test to compare the means occurring between individual treatments. P,0.05 was considered statistically significant. Author Contributions Conceived and designed the experiments: JH XL DH BL JC SL. Performed the experiments: JH XL. Analyzed the data: JH XL DH SL. Contributed reagents/materials/analysis tools: XL DH FE BL JC SL. Wrote the paper: JH XL BL JC SL. 5. Lewis GD, Figari I, Fendly B, Wong WL, Carter P, et al. (1993) Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother 37: 255–263. 6. Fendly BM, Kotts C, Vetterlein D, Lewis GD, Winget M, et al. (1990) The extracellular domain of HER2/neu is a potential immunogen for active specific immunotherapy of breast cancer. J Biol Response Mod 9: 449–455. 7. Kurai J, Chikumi H, Hashimoto K, Yamaguchi K, Yamasaki A, et al. (2007) Antibody-dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clin Cancer Res 13: 1552–1561. 8. Ghetie MA, Podar EM, Ilgen A, Gordon BE, Uhr JW, et al. (1997) Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells. Proc Natl Acad Sci U S A 94: 7509–7514. PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23270