99 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model Figure 3. Effects of the different PPAR-α agonists on peroxisomal protein levels in amino-acid restriction conditions. Organoids were grown in complete medium (Control) or in restriction medium for 96 hours (Starved), restriction medium with vehicle (DMSO) or (BSA), and in the presence of two different concentrations of LA (50 µM and 150 µM), WY-14643 (100 µM and 200 µM) or DHA (50 µM and 100 µM) (A, C, E) Representative immunoblot images (B,D,F) Peroxisomal protein levels relative to β-actin. Quantification of data shown in A, C, and E, respectively, and replicates thereof. Data represent mean ± SEM from 3 biological replicates (biological replicates are obtained from independent experiments) (*P < 0.05, **P < 0.01, ordinary oneway ANOVA with Tukey's post-hoc test). Effect of DHA on mitochondrial and peroxisomal proteome Since DHA had the largest rescuing effect on peroxisomal marker proteins, we set out to analyse the effect of this compound more comprehensively, using quantitative, targeted proteomics. Next to the above described peroxisomal protein panel, also previously validated panels of proteins involved in mitochondrial metabolism32, and glucose and glycogen metabolism39 were analysed. The results replicated the overall reduction of peroxisomal proteins upon amino-acid deprivation (Fig. 4a), and additionally showed a clear reduction of proteins involved in mitochondrial fatty-acid β-oxidation, TCA cycle and oxidative phosphorylation (Fig. 4b). The highest dose of DHA
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