Characterization of PEX19 Knockout Cells

All cell lines used in this study have been established and described previously (Schrul and Kopito, 2016 (link)). In brief, HeLa Kyoto wild-type cells served as parental cell line to generate PEX19 knock out cells (PEX19^−/−) using CRISPR/Cas9 technology. To generate cells that solely express non-farnesylated PEX19 (PEX19^−/−PEX19_C296S), PEX19^−/− cells were stably transfected with a plasmid encoding the PEX19_C296 mutant. Cells were cultivated at exponential growth rates in Dulbecco´s Modified Eagle Medium (DMEM) containing 4.5 g/L glucose and pyruvate (Gibco) and supplemented with 10% FBS (Biochrom) at 37°C and 5% CO_2. Cells were regularly tested for the absence of mycoplasma. To shift cells to catabolic conditions and to induce LD turnover, they were incubated in standard growth medium supplemented with 0.1% FBS (Biochrom) and in presence of the acyl-CoA synthetase inhibitor Triacsin C (Enzo) at a final concentration of 200 ng/ml for 18 h. To shift cells to anabolic conditions and to induce LD biogenesis, cells were treated with 200 µM oleic acid in complex with 0.2% BSA in standard growth medium for 18 h. Data reproducibility: All key findings of this manuscript with respect to the clonal PEX19-/-PEX19C296S cell line were reproducible in two different clones of this cell line (Supplementary Figure S3).