111 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model plates were filled with single double-positive cells to increase the chance of colonies growing. Sorted cells in 96-well plates were followed for the formation of single colonies with homogeneous shape for not longer than 12 days; wells with more than one colony were discarded. Seven colonies were followed and expanded under selection media with puromycin (2 μg/mL) and storage for further characterization. Measuring of autophagic flux by FACS Stable monoclonal-HuH7-3ΔG cells or enriched-LivOrg-3ΔG were grown in either control conditions (Ad. DMEM containing 1% P/S and 10% FCS) or in amino-acid restricted medium (a custom-made amino acid-free medium containing 1% P/S and 10% FCS). Organoids were treated with 150 nM of rapamycin to chemically induce autophagy. Bafilomycin A1 was used at 100 nM and Chloroquine at 50 µM. Cells were harvested using Accutase (Sigma, Cat N A9664-500ML) and individualized according with the methodology previously described. Cells were kept on ice in FACS buffer containing 1X HBSS, 2% FCS, 2% BSA. Cells were analyzed with the BD LSR-II cytometer (BD, Biosciences) equipped with a 488 nm laser and 561 nm laser. Samples were acquired using DIVA 8.0 software and at least 10.000 events for each sample were acquired. Data was saved as FCS 3.0 or 3.1 files and compensation was not required in any case. Data was processed with Kaluza software (Beckman Coulter). For calculating the autophagic activity, the GFP and RFP mean fluorescence intensity (MFI) (Geometric mean) was used to calculate the GFP/RFP ratio which reversely correlates with autophagic activity. Immunoblotting The protocol used for Western blotting was based on a previously published paper67. Ice cold Ad.DMEM/F12 (with or without amino acids) was used to collect organoids, and these were kept on ice for 10 minutes. Organoids were then centrifuged at 290G for 5 minutes and washed with PBS once. After a second centrifugation, they were reconstituted in 200 µL of radio immunoprecipitation assay buffer (1% IGEPAL CA-630, 0.1% SDS, and 0.5% sodium deoxycholate in PBS) supplemented with Complete Protease Inhibitor Cocktail (Cat. No. 1186145001; Sigma-Aldrich), Phosphatase Inhibitor Cocktail 2 (Cat. No. P5726; Sigma Aldrich) and Cocktail 3 (Cat. No. P0044; Sigma Aldrich). Organoids were then sonicated using a 30% amplitude four times for 10 seconds using Sonics Vibra cell VCX130 (Sonics & Materials inc.). Protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Scientific) and all the
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