97 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model Figure 2. Amino-acid restriction induces activation of autophagic flux in pMRX-IP-GFP-LC3RFP-LC3ΔG liver organoids. (A) Scheme of generation of pMRX-IP-GFP-LC3-RFP-LC3ΔG organoids. (B) Gating strategy for organoid sorting. First graph indicates the whole population. Second graph depicts the gating for single cells excluding aggregates. Third graph illustrates the separating based on GFP and RFP expression. (C) Representative brightfield and IF images of pMRX-IP-GFP-LC3-RFP-LC3ΔG liver organoids. Scale bar 100 µm. (D) Representative cytograms depicting the GFP and RFP intensities measured in the cells under different conditions. Wild type organoids (grey), organoids expressing pMRX-IP-GFP-LC3-RFP-LC3ΔG grown in complete amino acid medium (green), in amino acid restricted medium (blue), or in amino-acid restricted medium with bafilomycin A1 (maroon). Analysis was performed after 48 h (upper two graphs) or 96 h (bottom two graphs). (E) Left graph represents GFP/RFP fluorescence intensities ratio in organoids under control condition or in amino-acid restricted medium 48 h (left) or 96 h (right) and treated or untreated with 100nM bafilomycin. Data represent mean ± SEM from at least 3 biological replicates. Right graph shows GFP/RFP fluorescence intensities ratio in organoids under control conditions or in amino-acid restricted medium for 96 hours, and treated or untreated with bafilomycin. Data represent mean ± SEM from at least 3 biological replicates (*P < 0.05, **P < 0.01, one sample t test)
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