br The cell invasion assay was carried out by using
The cell invasion assay was carried out by using the BD BioCoat Matrigel™ invasion chambers with 8.0 μm pore PET membrane (BD Pharmigen TM, USA). Medium containing 10% FBS was added to the lower chambers and used as chemoattractant. MDA-MB-231 cells (2.5
× 105 cells/well) were resuspended in serum-free medium and treated or not treated (control cells) with BthTX-II (10 and 50 μg/ml). The cells were then seeded to the upper chamber, and let invade for 24 h, the non-invading cells were removed with a cotton-tipped applicator while invading cells were fixed, stained with Crystal violet and counted manually using a microscope at 10 X magnification in five different fields of triplicate membranes (EVOS â, AMG). The relative percentages of invasive cells were calculated by comparing to untreated cells (con-trol). All data represent mean ± SD of three independent experiments. The p value calculated when compared to the control. *p b 0.05 and **p b 0.01. NC: negative control.
2.15. Three-dimensional (3D) assay in matrigel
MDA-MB-231 and MCF10A cells were grown in a 3D culture by fol-lowing a previously described protocol  with minor modifications. Briefly, a 96-well plate was coated with a Matrigel™layer and incubated for 30 min at 37 °C in 5% CO2. After the Matrigel solidified, 3 × 104 cells were suspended in 100 μl of complete medium with 2% Matrigel and the mixture was added to the wells. Cells were treated with different con-centrations of BthTX-II (1, 10 and 50/ml) for 7 days at 37 °C and 5% CO2, and the development of spheroids was monitored. At day 7, pic-tures were taken at 10× magnification (Nikon Eclipse TS100). The assay was performed three times.
2.16. Integrin quantification
control monoclonal SC 79 as control. The samples were analyzed by the software BD Accuri (BD Accuri C6 – Biosciences, CA, USA).
2.17. E-cadherin, vimentin and CK5 expression by flow cytometry
MDA-MB-231 (3 × 106 cells/well) seeded onto 6-well plate for 24 h. MDA-MB-231 were treated with BthTX-II (10 and 50 μg/ml) for 24 h. In order to induce EMT in MDA-MB-231 cells, by EGF 30 ng/ml during 48 h. MDA-MB-231 were treated same conditions and analyzed (EGF-in-duced) and EGF-free cells . Briefly, cells were washed with PBS twice and harvested, fixed and permeabilized with BD Cytofix/ Cytoperm solution kit (BD Biosciences) at 100 μL per well for 20 min at 4 °C according to the manufacturer's recommendations and the cells blocked with 5% BSA for 30 min. Next, cells were incubated with antihuman CD324 E-Cadherin (BD-Biosciences), CK5 - EP1601y (Cell Marque) and anti-human vimentin antibodies (Sigma-Aldrich, Brazil) for 60 min at room temperature. Subsequently, cells were washed three times with PBS and incubated with anti-rabbit FITC (Thermo Fisher Scientific, Brazil) respectively, for 1 h. Cells were washed and an-alyzed by flow cytometry (BD Accuri-C6 Biosciences, CA, USA). The ex-periment was performed in triplicates.
2.18. Western blotting analysis
MDA-MB-231 cells, 24 h after treatment with BthTX-II (10 and 50 μg/ml), were harvested in solubilization buffer (Complete Lysis-M, EDTA-free, and protease inhibitor cocktail, Roche Diagnostics, USA) for total lysis. 10 μg of each protein extract was loaded onto reducing 10% (v/v) SDS-Tris-glycine polyacrylamide gels. For immunodetection, the separated proteins were then electrophoretically transferred onto Hybond nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden) according to Towbin (1979) . Membranes were blocked in TBST buffer (0.02 M Tris–HCl (pH 7.5), 0.15 M NaCl, and 0.1% Tween-20) containing 5% low fat milk, blotted for 2 h using anti-vimentin SAB4300676 (Sigma-Aldrich, Brazil) and beta Actin polyclonal antibody-BA3R at 1:200 (ThermoFisher Scientific, Brazil). The mem-branes were washed three times in TBST and PBS, and incubated for 60 min with a secondary antibody HRP-conjugated anti-rabbit igG (di-lution 1:2000 RAB0151 Sigma-Aldrich); Subsequently, the immunore-activity signals were visualized by autoradiography by using the chemiluminescent kit Amersham ECL Western Blotting Detection Re-agent and the Amersham Hyperfilm ECL (GE Healthcare Life Sciences). The intensities of the protein bands were quantified by using the soft-ware ImageJ, and the specific band-to-control ratio analyzed.
2.19. Statistical analysis
Statistical analyzes and graphs were obtained by using the Prism 5 software (GraphPad 5 Software Inc.), all experiments was performed three replicates. For the experimental analysis the t-test, One-way ANOVA and Two-way ANOVA followed by Tukey and Bonferroni post-test respectively, were used. Statistical significance was defined as *p b 0.05 (significant), ** p b 0.01 and ***p b 0.001 (highly significant). All the experiments were performed in triplicates.