Figure 7: Co-injection with DNase 1 prevents E. carinatus venom-induced tissue destruction.

(a) Western blot analysis of the appearance of H3Cit (top) in tail tissue homogenates taken 8 h after E. carinatus venom (LD₅₀) injection in the presence or absence of DNase 1. Quantification of H3Cit levels compared with H3 levels is shown (bottom). AU, arbitrary units; H3Cit, citrullinated histone 3; H3, histone 3. The data are presented as mean±s.e.m. (n=4). ***P<0.001 versus control mice; one-way analysis of variance, followed by Dunnett’s post-hoc test. The PVDF membranes were cut based on molecular weight of respective protein using protein molecular weight marker and then probed with respective antibodies. (b) Western blot analysis (left) of the appearance of E. carinatus venom in tail tissue homogenates taken 8 h after venom (LD₅₀) injection in the presence or absence of DNase 1. E. carinatus venom (20 μg) served as a positive control. The image of the corresponding Ponceau-stained PVDF membrane (right) shows E. carinatus venom, 20 μg (lane I), and equal protein loading of the tail tissue homogenates (lane II–IV); n=3. (c) Immunofluorescence images showing the accumulation of venom toxins from E. carinatus venom (LD₅₀)-injected mouse tail tissues sections in the absence (top row, middle) or presence of DNase 1 (100 U; top row, right). PBS-injected tail tissue served as a control (top row, left). The corresponding DIC images of the respective tissues are also shown (bottom row). Scale bars, 100 μm (n=3). (d) All the experiments in this group were performed simultaneously but the data are divided into five graphs (top three and bottom two) for clarity. Kaplan–Meier survival curves: E. carinatus venom, LD₅₀ (red line) in all the graphs; co-injection of E. carinatus venom (LD₅₀) with 25 U DNase 1 (blue line), 50 U DNase 1 (green line), 100 U DNase 1 (violet line), 100 U DNase 1 pre-incubated with 50 μM actin (grey line) and 100 U DNase 1 followed by ED AV (black line). ED AV, effective dose of antivenom. (n=10). *P<0.05, ***P<0.001 and NS, non-significant between groups using the Log-rank test. See Supplementary Fig. 5 for the effect of DNase 1 on the lethal potency of E. carinatus venom (LD).