Comparison of morphological data and quantitative data for three venoms that have varying effects on the cells.

(A) Morphological data represented by immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 24 hours exposure. Images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. 0.1% Triton T-100 was used as a positive control. Yellow stars represent wells in which activity was observed. Scale bar represents 200 μm. (B) Quantitative data of four cellular bioassays shown as bar graphs, with the activity of the venom represented relative to negative control (0 μg/mL). Live cell count (orange); cell surface area (grey); resazurin reduction activity (blue); ATP level (black). Increasing venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD; ‘*’ represents a statistically significant difference when compared to negative control, two-tailed test, p < 0.05 (Bonferroni-corrected).</p

Graphical overview of the bioassay workflow.

After injection of the venom, chromatographic separation by HPLC is performed, followed by high-resolution fractionation on 96- or 384-well plates for subsequent cellular bioassaying and protein identification using proteomics as described by Slagboom et al. [27]. Image created using www.biorender.com.</p

S8 Fig -

Bar graphs representing the time series data for the cell surface area in response to our panel of 10 snake species relative to the negative control (A) Time series data (3; 6; 12; 24; 48 hours) of six viper species at increasing venom concentrations (0; 1.2; 3.7; 11.1; 33.3; 100 μg/mL). (B) Time series data (3; 6; 12; 24; 48 hours) of four elapid species at increasing venom concentrations (0; 1.2; 3.7; 11.1; 33.3; 100 μg/mL). Venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD. (EPS)</p

Toxins identified by nano-LC-MS/MS following tryptic digestion of fractionated toxins from the venom of <i>N</i>. <i>mossambica</i>.

Abbreviations: prot_acc, protein accession number; prot_score, protein score; prot_cover, protein coverage; prot_desc, protein description; prot_seq, protein sequence; pep_seq, peptide sequence. (XLSX)</p

Brightfield (BF) and immunofluorescent microscopy images showing morphology of RPTEC/TERT1cells after 12 hours in presence of ten medically relevant snake species at increasing venom concentrations.

Both BF and fluorescent images were captured using confocal microscopy with 10X magnification. H342 staining is shown in blue, and PI in orange. All exposure settings were kept the same. The scale bar represents 500 μm. The images were scaled post-acquisition to the positive control. Yellow stars represent wells in which at least some activity was observed. (TIF)</p

Immunofluorescent microscopy images showing the staining of RPTEC/TERT1 cells with H342 and PI, which were used to quantify the total cell count.

Conditions shown for three different venoms: cytotoxic viper, cytotoxic elapid and non-cytotoxic elapid. (A) Image of cells stained with H342 (blue) and PI (orange) in the top row, with the lower set of images demonstrating the ability of the software to calculate all H342-stained cells (coloured dots). (B) Image of cells stained with PI alone, with the lower row showing the PI-stained cells as recognised by the software. Scale bar represents 200 μm. (EPS)</p

Overview of the 10 species included in the study with the proportion of the 12 major protein families in each venom (as percent of total venom).

Abbreviations: PLA2, phospholipase A2; SVSP, snake venom serine protease; SVMP, snake venom metalloprotease; LAAO, L-amino acid oxidase; 3FTx, three-finger toxin; KUN, Kunitz peptides; CRiSP, Cysteine-Rich Secretory Protein; NP, natriuretic peptide; %WV, percentage of venom. (DOCX)</p

Identification of bioactive fractions of <i>N</i>. <i>mossambica</i> venom (1 mg/mL) by correlating bioactivity data with proteomics data.

i: bioactivity chromatograms obtained by plotting the results of three bioassays: cell surface area (grey); resazurin reduction (blue); live cell count (orange). The peaks with negative minima indicate the presence of bioactive fractions; ii: Graphs representing the protein score chromatograms (PSCs), showing the individual venom proteins found with Mascot database searching of the digested well fractions. iii: UV traces of the snake venoms at 220 nm obtained by RP-HPLC. The vertical outer lines mark the bioactivity window, which includes the main activity peaks and their corresponding PSC peaks and RP-HPLC-UV chromatogram peaks. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD.</p

S7 Fig -

Bar graphs representing the time series data for the live cell count in response to our panel of 10 snake species relative to the negative control (A) Time series data (3; 6; 12; 24; 48 hours) of six viper species at increasing venom concentrations (0; 1.2; 3.7; 11.1; 33.3; 100 μg/mL). (B) Time series data (3; 6; 12; 24; 48 hours) of four elapid species at increasing venom concentrations (0; 1.2; 3.7; 11.1; 33.3; 100 μg/mL). Venom concentrations on the X-axes (in μg/mL) and percentage relative to negative control on the Y-axes. Measurements are presented as the mean of three individual experiments (N = 3), error bars depict SD. (EPS)</p

All raw data files, including raw bioassay data and proteomics data.

All raw data files, including raw bioassay data and proteomics data.</p