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  • br Western blotting analysis br


    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 DTT were then electrophoretically transferred onto Hybond nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden) according to Towbin (1979) [31]. 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.
    3. Results
    For assessing the antitumor and antimetastatic effects of BthTX-II on MDA-MB-231 cells, BthTX-II was firstly purified from B. jararacussu venom by CM Sepharose chromatography, resulting in 6 fractions (Sup-plementary Fig. 1A), and its homogeneity (CM-5 fraction) was con-firmed by reverse-phase chromatography (RP-HPLC) C18 (Supplementary Fig. 1B) and SDS-PAGE (Supplementary Fig. 1C).
    BthTX-II was cytotoxic to MDA-MB-231 cells in a dose-dependent manner (Fig. 1A) and showed cytotoxicity of around 50% at the highest doses. Interestingly, in the same concentrations, BthTX-II did not signif-icantly affect the viability of non-tumorigenic MCF10A cells. Next, we investigated whether the cytotoxicity induced by BthTX-II in MDA-MB-231 cells could be explained by the activation of autophagy. Inter-estingly, by evaluating autophagy through the fluorescent reagent, MDC, we observed that BthTX-II (50 μg/ml) was capable of inducing au-tophagy in approximately 70% of MDA-MB-231 cells and in about 30% of control breast cells (MCF10A) (Fig. 1B).
    We proceed evaluating the effects of BthTX-II (50 μg/ml) treatment on gene expression. BthTX-II was capable of modulating the expression of genes involved in the signaling apoptosis pathway (Fig. 1D). The re-sults showed that TNF and its receptor TNFRSF1A genes were upregu-lated by 6.26-fold and 8.42-fold, respectively. The transcriptional levels of the tumor suppressor genes BRCA1 and BRCA2 were respec-tively 3.79 and 1.42-fold increased upon treatment with BthTX-II. The BthTX-II stimulus induces the activation of apoptosis through upregula-tion of CASP8/TP53 of 1.91-fold and 2.42-fold, respectively. The MDM2 (E3 ubiquitin-protein ligase) and ANGPT1 (pro-angiogenic factor) were downregulated by 1.56 and 5.88-fold respectively. However, when compared to the control, BthTX-II treatment did not change sig-nificantly the expression of some genes involved in the apoptosis-signaling pathway, namely BAD, BAX, BCL2, BCL2L1, BIRC5, TNFRSF10B. The LDH activity was observed in MDA-MB-231 cells after treatment with BthTX-II (50 μg/ml) for 24 h, when compared to cells treated with Triton X-100 and with Control (untreated cells) (Fig. 1E).