Prism 6.0c

All data were analysed in Prism 6.0c (GraphPad Software Inc., San Diego, CA). Concentration response signalling data were analysed using a three-parameter logistic equation as described previously46 (link)47 (link). Signalling bias was analysed as described51 (link). Briefly, quantification of signal bias was performed using pharmacologically derived parameters of agonist affinity (Ka) and efficacy (tau) for each ligand in each of the three signalling pathways (cAMP accumulation, ERK1/2 phosphorylation, Intracellular Ca²⁺ mobilisation). The transduction ratio (tau/Ka) was extracted from standard concentration-response data that was analysed with the operational model of agonism (Kenakin & Christopoulos 2012). This value was used to calculate ΔΔ(tau/Ka) values through normalization of the transduction coefficient (tau/Ka) for each ligand in each signalling pathway to the reference ligand (hGLP1 in black) and the reference signalling pathway (cAMP). Data are presented on a log scale.
Statistical analysis was by One-way ANOVA (nonparametric) with Dunnett’s post test unless otherwise stated in the figure legends.

Prism 6.0c (ed. 2013, GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analyses. Student’s t-test was used to compare differences between means of the ELISA assay results. The following degrees of significance were established: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data are represented as means ± SD.

Statistical analyses were performed using GraphPad Prism 6.0c. All data shown represent the results obtained from triplicated independent experiments with standard deviation of the mean (mean ± SD). The sample sizes were chosen to allow for statistical significance testing assuming a major effect and a small variation. The variance was similar between the compared groups. The p values were calculated with two-tailed unpaired Student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons test or Bonferroni’s multiple comparisons test as indicated in corresponding figure legends. p < 0.05 were considered statistically significant.

Mice deficient in CatG (CatG^−/−, Ctsg^tm1Ley) in C57BL6 background (Christine Pham, Washington University, St Louis, MO, USA) were co-housed and age- and sex-matched C57BL/6J wild-type (WT) mice were used as controls. Animal studies were approved by the Cantonal Veterinary Office of Bern and conducted in accordance with the Swiss federal legislation on animal welfare. Mouse splenocytes were incubated with labeled antibodies against CD11c-PE (clone N418), CD45-PerCP (clone 30-F11), MHC-I-APC (clone AF6-88.5) (BioLegend, San Diego, CA, USA) and analyzed by flow cytometry on a FACSCalibur (BD Biosciences, Minneapolis, MN, USA). Relative mean fluorescence intensity (MFI) of MHC-I on dendritic cells (CD45⁺CD11c⁺) was determined using FlowJo (Tree Star, Inc., Ashland, OR, USA) and MFI of MHC-I from 4 mice/genotype was analyzed by Mann Whitney U test using Prism 6.0c (GraphPad. San Diego, CA, USA).

Data are shown as mean ± SD or mean ± SEM as appropriate. Statistical analysis was performed using Prism 6.0c (GraphPad, La Jolla, CA). For all experiments, 2-way analysis of variance followed by Sidak’s multiple comparisons test post-hoc analysis were used. The level of significance was set at p < 0.05.