*2.8. Protein–Protein Interaction Analysis*

STRING protein–protein interaction network analysis tool was used to evaluate protein–protein interactions identified among proteins with FC ≥ 1.5 from each cell line treated with *B. jararaca* venom. According to Doncheva and colleagues [114], STRING indicates interactions according to co-expression analyzes and evolutionary signals in all genomes based on data described in the literature between genes or proteins using functional classification systems such as Gene Ontology, KEGG (Kyoto Encyclopedia of Genes and Genomes), and Reactome.

Analysis of protein–protein interactions (PPIs) of proteins whose abundance increased more than 1.5× in the MCF7 cell line treated with 2.5 μg/mL of venom showed a high interconnection of proteins related to metabolic process (in red) and metabolism pathways (in blue) (Figure 5a). Interestingly, among the proteins identified in the MCF7, we observed clusters of highly connected proteins related to proteasome pathway (in green) and mRNA splicing (in yellow). We highlight the proteins PSMD2 and PSMD11 (26S proteasome non-ATPase regulatory subunit 2 and 11, respectively), PSME1 and PSME2 (Proteasome activator complex subunit 1 and 2, respectively), PSMC1, PSMC4, and PSMC5

(26S proteasome regulatory subunit 4, 6B, and 8, respectively), TXNL (Thioredoxin-like protein 1), and USP14 (Ubiquitin carboxyl-terminal hydrolase 14), which are all members of the proteasome pathway (in green) and proteasome complex, and, together with MST4 (Serine/threonine-protein kinase 26) and PSMD5 (26S proteasome non-ATPase regulatory subunit 5) they are all related to apoptosis (Figure 5a). PPI analysis of proteins identified in the MDA-MB-231 in the same conditions showed less interaction among the proteins identified with FC ≥ 1.5 (Figure 5b).

PPI analysis of the MCF7 cell lines with FC ≥ 1.5 treated with 0.63 μg/mL and FC ≤ 0.67 when cells were treated with 0.63 μg/mL and 2.5 μg/mL of venom are shown in Figure S6. Additionally, PPI analysis of proteins identified in the MDA-MB-231 at 0.63 μg/mL and 2.5 μg/mL venom treatment presenting FC ≤ 0.67 and FC ≥ 2.5 are shown in Figure S7.

Red: Biological Process of Metabolism. Blue: Metabolism Pathways (Reactome). Yellow: mRNA Splicing local network cluster (STRING). Green: Proteasome pathway (KEGG). Number of nodes: 125, Number of edges: 327, Average node degree: 5.23, Avg. local clustering coefficient: 0.47, Expected number of edges: 120, PPI enrichment *p*-value: < 1.0 × 10<sup>ƺ</sup>16.

(**a**)

**Figure 5.** *Cont*.

Red: Biological Process of Metabolism. Blue: Histone H3 complex (SMART, Simple Modular Architecture Research Tool). Green: apoptosis (Reactome pathways). Number of nodes: 31, Number of edges: 11, Average node degree: 0.71, Avg. local clustering coefficient: 0.387, Expected number of edges: 5, PPI enrichment *p*-value: 0.0192

(**b**)

**Figure 5.** Protein–protein interaction of proteins identified in (**a**) MCF7 and (**b**) MDA-MB-231 cell lines presenting FC ≥ 1.5 at 2.5 g/mL *B. jararaca* venom treatment.
