(5 mg/ml). The cultures were then re-incubated for an additional 4 h. After removal of the supernatant, 150 ml DMSO was added to each well to dissolve the crystals completely and then the absorbance was measured at 490 nm using an ELISA Reader (Bio-Rad, USA). Apoptotic cell morphology observation Cells were seeded in 24-well plates with glass slides on the bottom of wells, and treated with 50, 100, and 200 mg/ml of fucoidan for 48 h. After the slides were washed with cold PBS gently, cells were fixed by 4% polyformaldehyde for 1 h and then washed 3 times with PBS. The resulting cells were stained with 0.5 ml Hoechst 33258 (10 mg/ml) at 37uC for 10 min in the dark. The apoptotic features of cell death were established by staining cell nuclei with the DNA-binding fluorochrome Hoechst 33258 and assessing chromatin condensation by fluorescence microscopy (BX-50, Olympus, Japan) [31]. In each group, 5 microscopic fields were randomly selected and five hundreds cells were counted. Apoptotic cell was then calculated as the percentage of apoptotic cells over the total number of blue fluorescent protein-positive cells. Flow Cytometric Analysis The annexin V-FITC apoptosis detection kit was used for the apoptosis assay (Invitrogen). 4T1 cells (16106 cells/ml) were treated with 50, 100, and 200 mg/ml of fucoidan for 48 h. Cells were harvested by trypsinization, washed twice with PBS, and resuspended in 500 mL of binding buffer. Cell suspensions were then incubated with 5 mL of annexin V-FITC and 5 mL of propidium iodide (PI) for 10 minutes at room temperature in the dark. The cells were evaluated immediately by £ow cytometry (Coulter, Becton Dickinson, USA). Western blot analysis 4T1 cells were seeded in 6-well plates and treated with 50, 100, and 200 mg/ml fucoidan. After 48 h, with 2% SDS (10mM EDTA, 50 mM Tris base, 10% SDS, pH 8.0) and boiled at 100uC for 10 minutes. Protein concentrations were measured using the BCA protein assay. Briefly, 50 mg samples of protein were loaded on 12% SDS-PAGE gels, transferred to PVDF membranes and blocked with 5% nonfat milk in TBST buffer (20 mM Tris-HCl, 120 mMNaCl, 0.1% Tween for 1 hour [32]. Membranes were incubated with various primary antibodies against Bcl-2, Bax, Caspase-3, Survivin, VEGF, and phospho-p42/p44 extracellular signal regulated protein kinases (ERKs) at 4uC overnight. The rabbit polyclonal antibody for b-catenin, VEGF, and bcl-2, mouse monoclonal antibody for bax and ERKs, and rabbit monoclonal antibody for survivin were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA, USA). After washing, the blots were incubated with peroxidase-conjugated donkey anti-rabbit or anti- mouse secondary antibody for 2 hours. Protein expression was quantified with a Gel EDAS analysis system (Cold Spring USA Corporation) and Gel-Pro Analyzer 3.1 software (Media Cyber- netics). Assay for mitochondrial cytochrome C release This assay was performed according to cytochrome C releasing apoptosis assay kit’s (Biovision, USA) instructions. In brief, after treatment, 16106 cells were pelleted by centrifugation and washed twice with ice-cold PBS. Cell pellets were resuspended with 1 ml cytosol extraction buffer mix containing DTT and protease inhibitor, and incubated for 10 min on ice. After homogenization, unbroken cells and large debris were removed by centrifugation. Anticancer Effect of Fucoidan on Breast Cancer The resulting supernatants were saved as cytosolic extracts at 270uC. The pellets were resuspended with 100 mlextraction buffer mix containing DTT and protease inhibitor, and saved as mitochondrial fractions. Load 30 mg cytosolic and mitochondrial fractions isolated from 4T1 cells on 12% SDS-PAGE. Then western blot proceeded with cytochrome C antibody (Biovision). The same blot was reprobed for cytochrome oxidase (subunit II), a mitochondrial membrane protein, and it confirmed that the supernatant samples were free from mitochondrial contamination. Tumor growth in mice Female outbreed Balb/c mice at 4 weeks of age were purchased from Shandong Laboratory Animal Center. Seven mice were housed in wire-top cages with sawdust bedding in a clean, air- conditioned room at a temperature of 26uC and a relative humidity of 50%. 4T1 is a mammary carcinoma cell line that has been studied extensively [33]. In vivo generation of tumors was accomplished by injection of 56104 4T1 cells suspended in 50 ml PBS into the mammary fat pad. Animal body weight and tumor size were measured and recorded. Tumor size was measured every two days in two perpendicular dimensions (a = length, b = width) with a vernier caliper, and the size recorded as a volume (mm3) as calculated by a * b2/2. When tumors reached 8,10 mm2 in size, mice were treated by intraperitoneal injection of NS, 5 mg/kg.bw and 10 mg/kg.bw fucoidan every two days. A tumor growth curve was then constructed, and data were presented as mean 6 SE. After 20 days of tumor treatment, the mice were euthanized and their tumors were excised and weighed. The tumor specimens were fixed in 4% formaldehyde, embedded in paraffin, and cut in 4 mm sections for immunohistochemical analysis. For enumeration of pulmonary metastatic nodules, the metas- tases appeared as discrete white nodules on the black surface of lungs insufflated and stained with a 15% solution of India ink, and then bleached by Fekette’s solution. In situ TUNEL assay for apoptotic cells Apoptotic cell death in paraffin-embedded tumor tissue sections was examined using the TdT-FragEL DNA fragmentation detection kit (Calbiochem, San Diego, Calf, USA) according to the manufacturer’s protocol. Apoptotic cells were identified as dark brown nuclei under a light microscope. The number of apoptotic cells was counted 5 random fields (6400) in a blinded manner. Immunohistochemistry Immunohistochemical analysis of VEGF expression and MVD were performed according to the procedure described elsewhere [34]. The primary antibody is monoclonal rabbit anti-murine VEGF and monoclonal rabbit anti-murine CD34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). To quantify MVD, each slide was scanned at low power magnification (610–100). Two ‘hot spot’ areas with relatively higher number of new vessels were identified which were subsequently scanned at high power magnification (6400). Five random fields of each ‘hot pot’ area were analyzed. ELISA assays The concentration of VEGF in tumor tissues was determined using mouse VEGF ELISA Kit (Jingmei Biotech, Wuhan, China) according to the manufacturer’s instructions. The results of the ELISA assay in the tumor tissues were expressed as pg/mg. PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43483