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

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

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

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

Brightfield microscopy images of RPTEC/TERT1 cells in response to three different venoms, which were used to quantify the cell surface area.

Conditions shown for three different venoms: cytotoxic viper, cytotoxic elapid and non-cytotoxic elapid. Top row represents the ‘training’ of the software to differentiate cell populations (red dots) from the ‘empty well area’ (green dots) using a simple learn-by-example approach. The lower set of images provides the calculated cell surface area (depicted in red) and the ‘empty well area’ (in green). The scale bar represents 200 μm. (EPS)</p

Brightfield and immunofluorescent microscopy images showing the difference in morphology of RPTEC/TERT1 cells in presence of two venoms with contrasting effects.

(A) Cells exposed to 100 μg/mL E. ocellatus venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (B) Cells exposed to 100 μg/mL N. mossambica venom, with various channels (brightfield, Hoechst & PI) at 10X, 20X and 63X magnification. (C). Time series (0–150 min) of the effects of E. ocellatus venom (100 μg/mL), with a higher-magnification section (63X) clearly showing the monolayer detachment (60–70 min). (D). Time series (0–15 min) of the effects of N. mossambica venom (100 μg/mL) on cells, with a higher-magnification section (63X) showing the PI entering the cell (0–15 min). Scale bars lengths represented in the images.</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

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

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