2.13. Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology.24 All data are presented as mean ± SEM unless otherwise indicated. For in vitro assays (96‐well plate format) each individual experiment was performed in triplicate to ensure the reliability of the single value, and the average of n individual experiments was taken to represent each data point. For ex vivo and in vivo experiments, tissue isolated from a single animal or a single animal responsiveness represented n = 1.

The concentration response and dose‐response curves analyses were performed using GraphPad Prism version 7.04 (GraphPad Software Inc., La Jolla, California, USA). A four‐parameter logistic equation was fitted to the data and the pEC₅₀, pIC₅₀, t ₅₀ or ED₅₀ was calculated. The inclusion criterion was set at R ² > 0.900.

The possibility of linear correlation regarding potency for the 6 BoNT serotypes in a given pair of assays was assessed by calculating the Pearson's coefficient, using GraphPad Prism version 7.04 (GraphPad Software Inc.).

When assessing rank order of potency, a one‐way ANOVA followed by Bonferroni's post hoc test was performed for the in vitro assays (GraphPad Prism version 7.04, GraphPad Software Inc.). For the mouse ex vivo mPNHD assay, a linear regression was fitted to the data from each serotype (JMP Pro 13.0.0, copyright 2016, SAS Institute Inc, Cary, North Carolina, USA). Then, a predicted dose from a specified t ₅₀ time, 70 minutes, was calculated for all toxins, and the pmol/L potency was normalized to the least potent toxin, here serotype D, as described in Ref. 25. Comparison by Z‐test followed by Sidak correction was used for potency ranking (JMP Pro 13.0.0, SAS Institute Inc). The symbol “>” was used between datasets that revealed statistical significance and the symbol “=”was used between datasets showing no significant difference. The symbol “,” was used to indicate the rank order of potency in the in vivo experiments, for which statistical analysis was not performed (n = 1 ED₅₀ value calculated per serotype). In vivo BW data (see Tables S1 and S2) were analyzed by one‐way ANOVA followed by Dunnett's post hoc analysis (JMP 13.1.0., SAS Institute Inc). Statistical significance was established at P < 0.05 throughout the manuscript.