107 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model changed into differentiation medium (DM) containing: Advanced DMEM/ F12 (Gibco), supplemented with 10 mM HEPES, 1x GlutaMax, 1% PenicillinStreptomycin, 1x N2-Supplement, 1X B27-Supplement without vitamin A, 10 mM Nicotinamide, 1 mM N-AcetylCysteine, 100 ng/ml FGF-10, 50 ng/ml EGF, 10 nM Leu-gastrin, 50 nM A-83-01 (Axon Medchem) and 10 µM DAPT (Sigma). Between day 13 and 16, the medium was supplemented with 3 µM dexamethasone (Sigma Aldrich). These protocols were based on published literature59. Malnutrition in organoids using amino acid free medium In order to mimic the effects of a low protein diet, mature hepatic organoids were cultured in a custom-made amino acid-free medium as previously reported5. Organoids were kept in complete DM until day 12, when they were changed into restriction differentiation medium that contained all the same supplements but was completely depleted of all amino acids. Organoids were kept in starvation for 96 h until the end of day 16 when they were collected for further studies. In the case of the shorter (48 h) amino acid restrictions, organoids were kept in complete DM until day 14, when they were changed into restriction medium and collected at the end of day 16. Fluorescence microscopy Hepatic organoids were fixed with 1% formaldehyde in 0.1M PBS pH 7.4 for 15 min. Organoids were blocked and permeabilized in 0.1 M PBS with 20mM glycine, + 3% (w/v) BSA (9048-46-8; Fisher Scientific) and 0.1% (w/v) saponin (47036-50G-F; Sigma-Aldrich) pH 7.4 for 60 min at RT. Organoids were incubated with antibodies against PMP70 (Sigma: P0497) 1:400 in the same buffer. Subsequently organoids were washed in PBS three times and incubated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488 (A21206; Thermo Fisher Scientific, Invitrogen) at 1:800 dilution. For actin labeling, phalloidin Alexa Fluor 546 was used (A22283; Thermo Fisher Scientific) at 1:200 dilution in parallel with the labeling with secondary antibodies. Organoids were imaged in PBS containing DAPI. All steps were performed in a glass bottom 96 wells plate. Airyscan images were captured with a confocal microscope (LSM800; Carl Zeiss) equipped with a 32-channel gallium arsenide phosphide photomultiplier tube (GaAsP-PMT), Zen 2009 software (Carl Zeiss), and a 40 × 1.20 NA objective (Carl Zeiss). Peroxisome numbers per cell were quantified using FIJI. For each cell the grey values are adapted to visualize and quantify the separate peroxisomes because of the difference in staining intensities between the edge and the
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