*2.6. Principal Component Analysis*

PCA was applied to the differentially expressed proteins identified from both MCF7 and MDA-MB-231 cell lines based on the log2 FC of cells treated with low and high *B. jararaca* venom compared to the PBS treatment control group (Figure 4). The projection into the component space shows a distinct coordinated activity of proteins between the cell lines conditions. Orthogonal vectors show highly positive correlation between both venom concentrations in MDA-MB-231 cell line which may represent similar cell line responses to the different venom concentrations, but they present a highly negative correlation to both venom concentrations in MCF7 and respective set of expressed proteins. In the MCF7 cell line, however, we observe a negative correlation between the 0.63 μg/mL and 2.5 μg/mL venom treatment, indicating a differential response upon low and high dose venom treatment. PCA also shows differential correlation between both cell lines where the first two components showed 40% variation in the PC1 and 26.7% variation in the PC2 between the MCF7 and MDA-MB-231 cell lines. In addition, we observed clusters of proteins positively correlating with both low and high venom treatment in MDA-MB-231 cell line such as LAP3, H3F3B, and KRT1. On the other hand, we also observed proteins such as H2AC20 and TUFM correlating with low venom treatment, and proteins such as HEL-S-156an correlating with high venom treatment on MCF7 cell line (Table 1).

#### *2.7. Gene Ontology Functional Analysis*

The most enriched protein families and functional categories were analyzed based on highly abundant proteins with FC ≥ 1.5 for each cell line and treatment at low and high *B. jararaca venom* conditions (Figures S2–S5). The functional ontology classification analysis of these sets of proteins showed that both MCF7 and MDA-MB-231 venom-treated cell lines showed similar enriched categories. In addition, the most prominent enrichment was identified for treated cells with the sub-toxic dose of 2.5 μg/mL of venom. The molecular function enrichment analysis in both the MCF7 and MDA-MB-231 cell lineages showed an enrichment of proteins related to binding, structural molecule activity, and catalytic activity. In addition, the MCF7 cells had enriched, albeit in a lower amount, proteins related to function and transcriptional regulatory activity and carrier activity (Figure S2). The functional classification analysis related to biological processes showed enriched proteins related to the metabolic process and the cellular component organization or biogenesis. Moreover, the analysis of proteins identified in the MCF7 cell line presented proteins related to the cellular process, localization, biological regulation, stimulus response, developmental process, multicellular organismal process, and the immune system process (Figure S3). The analysis of protein distribution by cellular components showed an enrichment related to the "cell", protein complex, and organelle (Figure S4), and the enrichment analysis of protein family classification showed an enrichment of cytoskeleton proteins, ligase, nucleic acid binding, signaling molecule, modulating enzyme, calcium binding protein, and hydrase (Figure S5).

**Figure 3.** Hierarchical clustering of differentially expressed proteins detected in both MCF7 and MDA-MB-231 cells treated with low (0.63 μg/mL) and high (2.5 μg/mL) *B. jararaca* venom for 24 h. (**a**) Heatmap representation of the hierarchical clustering of proteins detected in both cell lines with quantification in at least two replicates showing the changes in protein abundance. The protein fold change is log2 transformed and normalized with mean-centering scale. (**b**) Protein Clusters extracted from the hierarchical clustering. X axis: Cell types treated with different *B. jararaca* venom concentrations (MCF7 0.63 μg/mL; MCF7 2.5 μg/mL, MDA-MB-231 0.63 μg/mL, MDA-MB-231 2.5 μg/mL); Y axis: mean-centered log2 Fold Change. Grey lines: individual proteins; Black line: average expression values per cluster.

**Figure 4.** Comparison of protein log2 Fold Change profiles across treated cell lines. Principal component analysis in a 2D graph represented by the first two components PC1 and PC2 explains 67.7% of the protein variability among the different conditions. Vectors that are closer are highly correlated. Vectors representing the conditions which are orthogonal or well-spaced in terms of the observed proteome indicate that those proteins can be closely related to each specific cell line condition.
