Triacsin c

BSA (essentially fatty acid free), FASN inhibitor (cerulenin), ACSL-1-5 inhibitor
(Triacsin C), ACSL-4 inhibitor (Troglitazone), Diacylglycerol acyltransferase
inhibitors (betulinic acid and A922500) and ACAT inhibitor (Sandoz 58–035) were
purchased from Sigma-Aldrich (St. Louis, MO). All fluorescent neutral lipid
analogs and labeling kits (S1 Fig) were from Invitrogen (Eugene,
OR).

YIC-C8-434 and triacsin C were obtained from Sigma-Aldrich and Enzo Life Sciences, respectively. Chemicals for lipid extraction (HPLC grade) were purchased from Fisher Scientific, and lipid standards were supplied by Avanti Polar Lipids with the exception of TAGs, MAGs, and free fatty acids, which were obtained from Sigma-Aldrich. The pJFH1 plasmid and LD540 were gifts from Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan) and Christoph Thiele (University of Bonn), respectively. Antibodies used to detect HCV core (rabbit antiserum 4210), E2 (AP33; a gift from Arvind Patel, Glasgow University), dsRNA (J2; supplied by SCICONS, Hungary), NS5A (sheep antiserum; a gift from Mark Harris, Leeds University), NS3 (mouse antibody; a gift from Thomas Pietschmann, TWINCORE, Hannover, Germany), human apoE (clone EP1374Y; supplied by Abcam), and human Perilipin-2 (PLIN2) have been described previously (25 (link), 41 (link)– (link)44 (link)). For detection of PLIN2 by indirect immunofluorescence, an antibody raised in guinea pig against the protein was used (Progen). Actin was detected in Western blots with an antibody purchased from Sigma.

For detection of mitochondrial ion O₂⁻ production, H9c2 myoblasts were stimulated at 37°C with 200 μmol/l palmitate-BSA in the absence or presence of MCP (0.01%). Thereafter, cells were washed and loaded with 5 μmol/l MitoSOX^TM Red for 30 min, at 37°C. The fluorescent signal was analyzed by recording FL2 fluorescence in a Gallius^TM flow cytometer (Beckman Coulter).
To evaluate the mitochondrial transmembrane potential (ΔΨm), H9c2 myoblasts were incubated with 4 μmol/l of Rhodamine 123 for 15 min at 37°C. Stained cells were washed with serum-free medium and stimulated with the indicated doses with palmitate-BSA for 24 h at 37°C. After treatment, cells were washed with PBS and changes in fluorescence were monitored using flow cytometry analysis. In some experiments, before incubation with 200 µmol/l of palmitate-BSA, H9c2 cells were pretreated for 30 min with 3 µmol/l of Triacsin C, an inhibitor of long fatty acid acyl-CoA synthetase (Sigma, St Louis, MO) or with the indicated dose of MCP. Experiments were repeated at least three times. Cells were also visualized on a Leica TCS SP5 X confocal microscope with a ×40 objective.

Collagenase IV, fatty acid-free bovine-serum albumin (BSA), OA sodium salt (≥99% purity), Triacsin C (long-chain acyl-CoA-synthetase inhibitor), and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA); BODIPY 493/503 from Invitrogen (Buenos Aires, Argentina); silica-gel-G–precoated 20-x-20-cm thin-layer-chromatography plates from Merck (Buenos Aires, Argentina); and lipid standards from Nu-Chek Prep, Inc. (Elysian, MN, USA). All chemicals and solvents were of analytical and HPLC grade, respectively.

MMV019719, MMV665924, and MMV897615 were freely available as part of the Medicines for Malaria Venture’s (MMV) Malaria Box. Triacsin C was purchased from Sigma-Aldrich (St. Louis, MO, ref. T4540). The 3D7 P. falciparum strain was originally obtained through MR4 as part of the BEI Resources Repository, NIAID, NIH: 3D7, MRA-102, deposited by D. J. Carucci. We also used 3D7- IG06 (a fast-growing 3D7 clone)^{64 (link)} donated by Daniel Goldberg and Dd2-Polδ²³ donated by Marcus Lee. The Malawi parasites (CF04.008 10B and CF04.009 6D and 1 G, two different patient isolates that were subcloned after culture adaption) were kindly donated by Danny Milner^{26 (link)}. Parasites were cultured by standard methods^{65 (link)} in RPMI 1640 medium supplemented with 28 mM NaHCO₃, 25 mM HEPES, 400 μM hypoxanthine, 25 μg/mL gentamicin, and 0.5% AlbuMAX II (Life Technologies, Carlsbad, CA 11021-045). The human erythrocytes were sourced ethically, and their research use was in accord with the terms of approved protocol.