Triacsin c

Each LD homeostasis inhibitor used was diluted in DMSO based on manufacturer’s instructions and optimum inhibitor concentration was determined based on 100% host cell viability determined by trypan blue staining, and changes in LD numbers per cell. The optimum concentrations determined for each inhibitor was: Triacsin C (Enzo Life Sciences, Farmingdale, NY, USA)– 10 µM, T863 (Sigma-Aldrich)– 10 µM, CAY10499 (Cayman Chemicals, Ann Arbor, MI, USA) - 10 µM, Atglistatin (Cayman Chemicals)– 20 µM.

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).

Compound C (Cayman Chemicals) was suspended in dimethyl sulfoxide (DMSO) at a stock concentration of 10 μM and added to J774A.1 macrophages at the indicated concentrations 1 h prior to bacterial inoculation. The Compound C dosage was determined based on data from previous studies (50 (link)). Compound C remained on macrophages throughout infection. The cytotoxicity of Compound C in J774A.1 macrophages was determined using a Vybrant 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell proliferation assay kit (Thermo Fisher) according to the manufacturer’s protocol. Atglistatin (30 μM) (Cayman Chemicals), triacsin C (10 μM) (Enzo Life Sciences), and T863 (10 μM) (Cayman Chemicals) were suspended in DMSO (12 (link), 30 (link)). For lipid inhibitor studies, we pretreated cells overnight with compounds and continued treatment throughout infection.

The primary antibodies against phosphorylated (p)-Akt (Ser473), p-ACC (Ser79), p-AMPK (Thr172), Akt, ACC, AMPK, and ACSL1 were purchased from Cell Signaling Technology (Tokyo, Japan). Anti-β-actin was obtained from Sigma-Aldrich (MO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (PA, USA), and Triacsin C was obtained from Enzo Life Sciences (NY, USA).

[³⁵S]-methionine and [³H]-palmitate were purchased from Perkin Elmer Life Sciences (Boston, MA). [¹⁴C]-labeled protein molecular weight markers and Mitotracker Red CMXRos were purchased from Invitrogen (Carlsbad, CA). Protran nitrocellulose membranes were obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). 5-(Tetradecyloxy)-2-furoic acid (TOFA) and triacsin C were obtained from Enzo Life Sciences. C75, 2-bromopalmitate, trimetazidine, rifampicin, fatty-acid free bovine serum albumin, sodium palmitate, oleic acid, glucose, glutamine, oxaloacetate and dimethyl-α-ketoglutaric acid were obtained from Sigma (Saint Louis, MO). Etomoxir was obtained from Tocris Bioscience (Bristol, UK).