95 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model Figure 1. Longer amino-acid restriction lead to more severe loss of peroxisomal proteins and induces the formation of autophagosome-like structures. (A) Representative immunoblot images. (B) Protein levels relative to β-actin. Quantification of data shown in A and replicates thereof. For ACOX1 quantification, antibody intensities of both bands were summed. Organoids were grown in complete medium (control) or in amino-acid restricted medium for 48h or 96h. Data represent mean ± SEM from 3 biological replicates obtained from independent experiments (*P < 0.05, **P < 0.01, ordinary one-way ANOVA with Tukey's post-hoc test). (C) Heatmap representing targeted proteomics data from control conditions, 48 h of amino-acid restriction and 96 h of amino-acid restriction. Data represents 4 biological replicates. (D) Representative single Airyscan slice immunofluorescence images. On the left side control organoids and on the right organoids after 96 hours in low amino acid conditions. Peroxisomes stained for PMP70 in green, with DAPI nuclei counter stain (blue) and phalloidin (red). Scale bar, 10 μm. (E) Quantification of number of peroxisomes per μm2 per cell based on PMP70 staining in D from 3 biological replicates (*P < 0.05, **P < 0.01, **P<0.001 ordinary one-way ANOVA). (F) Electron microscopy images upon 48 and 96 hour amino-acid restricted organoids showing double-membrane electron-dense structures. N; nucleus. (G) Relative gene expression of peroxins in organoids in control condition or in amino-acid restricted for 96 hours. Data represent mean ± SEM from 3 biological replicates (*P < 0.05, **P < 0.01, unpaired t-test).
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