The software identified in the jararhagin-treated Src inhibitor HUVECs 59 up-regulated genes with fold changes greater than 1.5 and p values < 0.05 and 11 down-regulated genes with fold changes greater than −1.5 and p values < 0.05 compared to un-treated cells. Analyzing the results according to the inflammatory response induced by jararhagin on HUVECs, among these 59 up-regulated genes, 25 were related directly or indirectly with

the inflammatory response. Down-regulated genes with fold changes greater than −1.5 were detected in 7 genes corresponding to inflammatory mediators (the complete abbreviations and acronyms of each gene is shown in Table 1). Jararhagin up-regulated the expression of 14 important genes

involved in cell signaling and cell–cell interaction: E-selectin, VCAM-1, IL-8, IL-6, THBD, SULF1, CXCL-6, ANGPT2, CDKN1B, DTR, DAF, TLN1, CSF2RBIL, IL1RL1 (respective fold changes were 5.33; 2.75; 2.23; 1.97; 1.97; 1.95; 1.91; 1.88; 1.82; 1.75; 1.66; 1.64; 1.59; 1.54). Another gene group up-regulated by jararhagin is related to cell death, with expression of 20 genes: CD69, SAT, VCAM-1, IL-8, CEPBD, IL-6, THBD, SULF1, ANGPT2, CDKN1B, SOD2, DTR, PEG10, ARG2, GULP1, DAF, CSF2RB, ILRL1, BTG1, SH3BP5 (respective fold changes were 3.39; 3.23; 2.75; 2.23; 2.15; 1.97; 1.97; 1.95; 1.88; 1.82; 1.78; 1.75; 1.74; 1.69; 1.67; 1.66; 1.59; 1.54; 1.54;

1.53). A total of 10 Ibrutinib genes involved with inflammatory diseases were up-regulated by jararhagin: E-selectin, SAT, IL-8, CEBPD, IL-6, SOD2, MMP-10, ARG2, DAF, IL1RL1 (5.33; 3.23; 2.23; 2.15; 1.97; IDH activation 1.78; 1.73; 1.69; 1.66; 1.54). Real time-PCR was utilized to obtain the time-course of expression and quantitation of 8 genes from those up-regulated in microarray analysis. We chose representative genes in each one of the biological effects cited above: E-selectin, VCAM-1, IL-8, IL-6, CXCL-6, ANGPT2; CD69, VCAM-1 and MMP-10. The RNA was extracted from HUVECs at 3 different time-points (3, 6 and 24 h) after the treatment with jararhagin (200 nM). The cDNA was transcribed and quantified by real time PCR using the relative quantification method (2−ΔΔCT2−ΔΔCT). We performed the treatment of HUVECs with LPS 1 μg/mL as a positive control in our experiments, indicating that the cells were responsive and our sample was completely depyrogenated. LPS was used as positive control for our experiments once we selected genes for inflammatory response and it stimulates leukocyte and blood endothelium through the LPS recognition systems, binding with CD14 and transferring to TLR4 and MD-2 complex, resulting in the production of downstream inflammatory cytokines and leukocyte adhesion molecules by the activation of NF-κB and AP-1-dependent transcriptional pathways (Sawa et al.

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