113 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model Isotopically-labelled concatemer derived standard peptides were selected for the targets of interest (QconCAT technology, PolyQuant GmbH). In-gel digestion, LC-MS and analysis were performed as previously described by Wolters et al32. The list of peptides used for targeted proteomics of peroxisomal proteins can be found in Supplementary Table 1. Branched chain fatty acids measurements Organoids were incubated with 25 µM phytol (Sigma Aldrich) for 48 h prior to quenching with ice-cold methanol. Methanol was then evaporated under a constant stream of N2.The remaining pellet was reconstituted in PBS and using a 30% amplitude for four times for 10 seconds using Sonics Vibra cell VCX130 (Sonics & Materials inc.). Samples were further processed as previously described5. Statistical Analysis Results are expressed as mean ± standard error of the mean (SEM). Biological replicates are considered as independent experiments using independent organoid lines. Analyses were performed using GraphPad Prism Software Version 9.02 (Graphpad Software). Statistical significance between comparisons is provided in figure’s legends. no indication means no significant changes (ns). FUNDING This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 812968, and by the Netherlands Organisation for Health Research and Development under grant number 435005013. ACKNOWLEDGEMENTS We would like to thank Niels Kloosterhuis and Marieke Smit for their technical support with animal work. We would like to thank Nicolette Huijkman for the lentivirus production and establishment of stable cell lines. We would also like to thank Tjaša Košir for her advice on the choice of PEX transcript and interpretation.
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