101 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model Figure 4. DHA supplementation prevents the loss of multiple peroxisomal and mitochondrial proteins and prevents autophagy in amino-acid restricted (96 h) organoids. Organoids were grown in complete medium (Control) or in amino-acid restriction medium for 96 h (Starved) with DMSO or DHA (50 µM and 100 µM) (A) Heat map of peroxisomal proteins detected including several metabolic pathways and transporters quantified using targeted proteomics. (B) Heat map of mitochondrial proteins detected including fatty acid oxidation, TCA cycle, ETC and reactive oxygen species detoxification quantified using targeted proteomics. Z-scores were used to normalize the data and protein abbreviations are used according to Uniprot. (In red high Z-score indicating upregulated proteins and in blue low Z-score indicating downregulated proteins). (C) Triglycerides levels in control, starved (96 hours) and starved treated with DSMO (vehicle) and 50 and 100uM of DHA (as per color legend). Data represents 6 biological replicates from independent experiments ± SEM (*P < 0.05, ordinary one-way ANOVA with Tukey's post hoc test). (D) Scheme of the metabolism of phytol into phytanoyl-CoA and pristanoyl-CoA in the peroxisome. (E) Concentration of phytanic and pristanic acid in the supernatant of organoids. Data represents 3 biological replicates ± SEM (*P < 0.05, **P < 0.01. RM One-way ANOVA with Tukey's post-hoc test). (F) GFP/RFP fluorescence intensities ratio in Huh7 cells in control conditions and 48 h of amino-acid restriction treated and untreated with DMSO or DHA (50 and 100 μM). Data represent mean ± SEM from 3 biological replicates (*P < 0.05, **P < 0.01 One-way ANOVA with Tukey's post-hoc test). (G) GFP/RFP fluorescence intensities ratio in organoids expressing pMRX-IP-GFP-LC3-RFP-LC3ΔG. Data represent mean ± SEM from 3 biological replicates (*P < 0.05, **P < 0.01 One-way ANOVA with Tukey's post-hoc test ).
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