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in order to unveiling molecular pathways involved in AgNPs-EPSaer cytotoxicity. A total of six gels were run to achieve a statistically significant measure of the differences in protein expression between the analyzed groups. Protein spots were quantified, normalized and inter-gel matched and only differentially expressed proteins with the threshold settings with at least fold change ≥ 1.3 and p-value < 0.01, were considered regulated (Supplementary Table 1). Figure 6A shows a prototype of proteomic map of SKBR3 cells with 253 labels corresponding to the protein spots showed a significant difference. Among the differentially expressed spots (Figure 6B), 202, corresponding to 80%, were selectively modulated in AgNPs-EPSaer treated cells, only two proteins spots were modulated selectively in AgNPs-EPSanaer and only two in EPS free metal treated cells, confirming the prominent biological activity of AgNPs-EPSaer. Overlapping modulations are shown, also. The trend of up and down regulated proteins compared to untreated cells are shown in Figure 6C. Protein identification was performed by MALDI TOF-MS/MS analysis. We successfully identified 118 protein spot, corresponding to 83 proteins (Supplementary Table 2). Several proteins were found in different isoforms and then detected in multiple spots. In order to construct protein-protein interaction networks regulated by AgNPs- EPSaer we used STRING database [44]. Figure 7A depict the interaction networks of the AgNPs-EPSaer regulated Figure 4: Effect of AgNPs-EPS treatment on intracellular ROS generation. ROS were measured using DCFH-DA (A) and DHE (B) and quantified through spectrofluorometry. Data were normalized for cell number determined by MTT assay and expressed as percentage in respect to the untreated cells used as control. Statistical significance was assessed by the Student’s t-test: *p < 0.05 was considered significant, **p < 0.01 highly significant and ***p < 0.001 very highly significant. The data are expressed as mean value ± SD. www.impactjournals.com/oncotarget 9690 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
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