Figure 9: DNase activity of N. naja venom is essential for degrading NETs and increasing its lethal potency.

(a) N. naja venom-stimulated NET formation was quantified using MPO-DNA capture ELISA in both dose- (top) and time-dependent (bottom) assays. US, unstimulated cells. The results are expressed as the percent increase relative to the US; mean±s.e.m. (n=6). *P<0.05, **P<0.01, ***P<0.001 versus the US; one-way analysis of variance, followed by Dunnett’s post-hoc test. PMA (50 nM) served as a positive control. (b) Western blot analysis (left) of the appearance of N. naja venom in tail tissue homogenates taken 8 h after venom (LD) injection in the presence or absence of actin (50 μM). N. naja venom (20 μg) served as a positive control. The image of the corresponding Ponceau-stained PVDF membrane (right) shows N. naja venom, 20 μg (lane I) and equal protein loading of tail tissue homogenates (lane II–IV); n=3. (c) Representative immunofluorescence images were captured 8 h after N. naja venom (LD) injection in mouse tails and did not show an accumulation of venom (top row, middle), similar to the PBS-injected control tissue (left). However, N. naja venom accumulated when the venom (LD) was pre-treated with 50 μM actin before injection. The corresponding DIC images of respective tissues are shown (bottom). Scale bars, 100 μm (n=3). (d) Kaplan–Meier survival curves after injections of N. naja venom (LD, red line) or N. naja venom (LD) pre-incubated with 50 μM actin (blue line); n=10. ***P<0.001; Log-rank test.