Anne-Marie Koop

160 Bioinformatics and statistical analysis Quantative data (except the lipidomic data, see below) are expressed as mean±standard error of the mean (SEM). GraphPad Prism 5.04 was used for data analysis. Comparisons between control and PAB-groups were tested with unpaired Students t-test, whereas comparisons over time (2 versus 5 weeks, 5 versus 12 weeks and 2 versus 12 weeks) within groups (control or PAB) were tested with one-way ANOVA. Bonferroni post hoc correction was used for multiple testing. The p-value of <0.05 was considered as statistical significant. Control groups were pooled, since no differences were observed in time. With respect to the measurements of the mitochondrial respiration, control groups were presented individually. Bioinformatics and statistical analyses of the lipidomics data were performed as described before [32]. Totals for the major classes were defined as the summation of the relative abundance of all identified lipids within the same class normalized to the corresponding internal standard. Statistical analysis of the lipid classes were performed using the statistical programming language R (https:// www.r-project.org/ ) together with the “MixOmics” package (https://doi.org/10.1371/ journal.pcbi.1005752). A q-value of 0.01 was assumed to be significant. Partial least squares-discriminant analysis (PLS-DA) was used to assess the variable importance in the projection (VIP)-score of individual lipids. Lipids were assumed to be significant if p < 0.05, false discovery rate (FDR) < 0.1 and VIP > 1. Boxplots displays the full range (minimum, first quartile, median, third quartile, and maximum), including statistical outliers. Assessment of inflammatory status and oxidative stress RV gene expression of different inflammatory and oxidative stress markers were assessed at mRNA level by standard qPCR. Macrophage infiltatrion in the RV was assessed by cluster of differentiation 68 (CD68) staining, as decribed previously. 34 Advanced oxidation protein products (AOPP) (ab242295, Abcam, Cambridge, United Kingdom) and anti-oxidant capacity assay (ab65329, Abcam, Cambridge, United Kingdom) were performed in RV tissue to the manufacturer’s instruction. Plasma levels of growth differentiation factor 15 (GDF-15) were measured by ELISA (MGD150, R&D, USA). AOPP were assessed in plasma as well (ab242295, Abcam, Cambridge, United Kingdom).