46 Chapter 2 Amino acid deprivation reversibly reduces mitochondrial and peroxisomal content in intestinal organoids In view of the key role of mitochondria and peroxisomes in intestinal homeostasis26,41, we examined the effect of amino acid deprivation on these organelles in intestinal organoids. In contrast to hepatic organoids, amino acid starvation did not affect the protein level of PGC1-α in intestinal organoids (Figure 6a,b). The levels of the peroxisomal proteins PMP-70 and catalase were, however, reduced in starved intestinal organoids (Figure 6a-b). Figure 6. Impact of amino acid starvation, re-supplementation and rapamycin on mitochondria and peroxisomes in intestinal organoids. (A) Representative immunoblot images. (B) Quantification of data shown in (A). Protein levels were normalized to total protein (see methods section). Organoids were grown in complete culture medium (control), amino-acid-free medium (starved), amino-acid-free medium for 48 h followed by complete culture medium for 48 h (re-supplementation) or in amino-acid-free medium for 48 h supplemented with 2nM rapamycin. Data represents mean of 3 biological replicates (from independent experiments) ± SEM (*P<0.05, ** P< 0.01, *** P<0.001, Two-way ANOVA with Tukey’s post-hoc test). (C) Relative intensity of total protein measured in each entire lane (used for total protein normalization as explained in methods). Individual data points are shown for each biological replicate. (D) Mitochondrial copy number expressed mitochondrial DNA normalized to β-globulin as reference. Data represents mean of 3 biological replicates (from independent experiments) ± SEM. (*P<0.05, ** P< 0.01 Ordinary one-way ANOVA).
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