Prism v6

All values presented in the results section are the mean or mean ± standard deviation of the mean of three independent analyses, calculated using GraphPad Prism v6 Software (GraphPad Software Inc., La Jolla, CA, USA). Interpolated sigmoidal curves (4-parameter logistic curve, 4 PL) were determined automatically using GraphPad Prism v6 Software (GraphPad Software Inc., La Jolla, CA, USA).

GTPγ[³⁵S]binding assay was performed in quadruplicates. Dose–response curves were calculated with GraphPad Prism v 6.0.7 using the log(dose) response curve with variable slope. BRET assay was performed in triplicates. Association kinetics was calculated with GraphPad Prism v 6.0.7 using monophasic association. Data are mean ± SD calculated from three independent experiments (SI Tables S1–S6).

IC50 calculations and the respective curves were generated by GraphPad Prism v6.0 Software. The effect of the interaction of different agents on combination treatment (synergistic, additive, antagonistic) was analyzed with the freeware program “Synergy” based on the R statistical language (R v2.13.1) as published elsewhere [49 (link)]. For each interaction-effect experiment, the Chou-Talalay method [50 (link)] was primarily used and results were verified with the Plummer method [51 (link)]. For comparisons of treatment groups, unpaired t-tests (Mann-Whitney), paired t-tests, and one-way or two-way ANOVA (where appropriate) were performed. For ANOVA, Bonferroni posthoc analysis was used to compare treatment groups. All analyses were performed with GraphPad Prism v6.0. Differences with a P value ≤ 0.05 were considered significant.

MicroRNA expression was assessed using SDS2.4 software (Life Technologies) and fold change was determined as for comparative Ct method (26 (link)). Statistical analyses were carried out using SPSS v19.0, Sigmaplot v13 or GraphPad Prism v6.0. Differences between groups were assed using the independent t-test, one-way ANOVA, or one-way ANOVA repeated measure analysis. Following a significant ANOVA result, differences between pairs of groups were detrmined via the Tukey post-hoc test (Prism v6.0). Receiver operating characteristic (ROC) analysis was performed using marker expression on a continuous scale as the test variable and disease status as the state variable (Sigmaplot v13). For the ROC analyses, pre-test prior-probability was set to 0.5 and Cost Ratio to 1.0 (27 (link)). The optimal cutoff value to dichotomise microRNA expression was computed from sensitivity and specificity using the slope m by finding the cutoff that maximizes the function: sensitivity-m(1-specificity) (Sigmaplot v13) (28 (link)). The accuracy of the test was defined by the area under the curve (AUC), whereby AUC = 0.5 means no diagnostic ability and AUC = 1 means perfect diagnostic ability.

All graphs were generated in Prism v6.0. Virus infectivity curves were generated and Area Under the Curve was calculated in Prism and used for comparisons of neutralization activity. Neutralization IC50 titers were also calculated in Prism from the virus infectivity curve using log transformation of x-values, normalization of y-values, and linear regression of dose-response inhibition with variable slope. A Mann-Whitney test or Wilcoxon matched pairs analysis was used to compare two groups, while a Kruskal-Wallis test was used for to compare more than two groups. A non-parametric Spearman’s test was used to assess correlations between variables in Prism v6.0. P values less than 0.05 were considered to be significant.