O1008

The incorporation of acyl-CoA was measured with a FRET-based assay. The reaction was mixed in a 50 µl volume containing 0.1 µM Faa1-WT-mCherry or Faa1-4D-mCherry, 60 µM Atg9-vesicle like liposomes containing lissamine rhodamine-DHPE (L-1392; Invitrogen) and NBD-DPPE (810144C; Avanti Polar Lipids, Inc.), 2.7 mM oleic acid (O1008; Sigma-Aldrich), 1 mM ATP (A2383; Sigma-Aldrich), 0.1 BSA (A9647; Sigma-Aldrich), and 10 mM MgCl₂ in buffer containing 200 mM NaCl and 25 mM Hepes pH 7.5. The reaction was started with the addition of 0.05 mM CoA (C3144; Sigma-Aldrich) or the same volume of buffer for the “−CoA” samples. Fluorescence intensity with an excitation wavelength of 485 nm and an emission wavelength of 535 nm was measured with a Tecan SPARK Multimode Microplate Reader (RRID:SCR_021897; Tecan Life Sciences) before the initiation of the reaction and 30 min after initiation while incubating at 30°C. For normalization, the measurements were divided by the measurement before initiation. Further, the fluorescence intensity of inactive Faa1 (“−CoA”) was subtracted from the one of active Faa1 (“+CoA”) to receive the increase in fluorescence intensity.

We prepared the oleic acid (OA) solution as described in [53 (link)]. To summarize, we dissolved the powder OA (O-1008 Sigma-Aldrich, Germany) at a final concentration of 12 mM in phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and pH 7.4) containing 11% fatty acid free bovine serum albumin (FFA-BSA; 0215240110, MP Biomedicals, Santa Ana, CA, USA). The solution was then sonicated and shaken overnight at 37 °C with an OM10 Orbital Shaking Incubator (Ratek Instruments Pty, Ltd., Boronia, Australia). The OA solution was filtered with a 0.22 μM filter, aliquoted, and kept at 4 °C. We utilized a fresh aliquot for each experiment.

Huh-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, in a humidified atmosphere containing 5% CO₂ at 37 °C. For experimental purposes, cells (2–3 × 10⁶) were seeded in 100 mm culture dishes and, after 24 h, the culture medium was carefully removed and cells were washed twice with phosphate-buffered saline (PBS). Then, cells were treated for 16 h with 400 µM fatty acids (PA or OA), or Tg (2.5 nM), or SCD1i (5 µM) or vehicle (0.2% ethanol + 0.01% DMSO). Palmitic acid (P5585, Sigma-Aldrich) and oleic acid (O1008, Sigma-Aldrich) were dissolved in ethanol at a stock concentration of 148 mM; final concentration of ethanol was 0.2% in the medium. Tg (T9033, Sigma-Aldrich) and SCD1i (sc-205109A, Santa Cruz Biotechnology) were dissolved in DMSO at a stock solution of 2 mM and 25 mM, respectively; final concentration of DMSO was ≤ 0.01% in the medium. All treatments were carried out in serum-free medium containing 0.1% free fatty acids-Bovine Serum Albumin. Media were collected to isolate EVs by a differential centrifugation^{26 (link),45} (see below). Cells were recovered and counted by Countess TC20 automated cell counter (Bio-Rad). Cell viability was assessed by Trypan Blue stain exclusion.

Palmitic acid (PA; P0500, Sigma-Aldrich) and oleic acid (OA; O1008, Sigma-Aldrich) were dissolved in 100% ethanol and conjugated to 10% fatty acid-free bovine serum albumin (BSA) (A0281, Sigma-Aldrich) in RPMI 1640 medium (HyClone). The molar ratio of PA/OA:BSA is 6:1. The primary fibroblasts were treated with PA or OA at a concentration of 500 μM for 24 h.

The oleic acid (OA) was prepared as in [36 (link)] and no diluent was used to dissolve it. In brief, we dissolved the powder OA (O-1008 Sigma-Aldrich, Taufkirchen, Germany) at a final concentration of 12 mM in phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and pH 7.4) that contained 11% fatty acid-free bovine serum albumin (FFA-BSA; cat#:0215240110, MP Biomedicals, Santa Ana, CA, USA). The solution was then sonicated and shaken at 37 °C overnight using an OM10 Orbital Shaking Incubator (Ratek Instruments Pty, Ltd., Boronia, Australia). The OA solution was filtered using a 0.22 µm filter, aliquoted, and stored at 4 °C. We used a fresh aliquot for each experiment.

Nineteen healthy Border Leicester Cross ewes, aged between 1 and 3 years and weighing 52 kg (47–54), were randomly assigned to one of three groups; injury by intravenous infusion of oleic acid (OA, n = 7), by OA and intratracheal E. coli lipopolysaccharide (IT, n = 7), or by OA and intravenous E. coli lipopolysaccharide (IV, n = 5).
Briefly, animals were anesthetized with ketamine, midazolam, and fentanyl. Continuous neuromuscular blockade was maintained by infusion of vecuronium. After induction, animals were tracheostomized and ventilated using a low tidal volume strategy. After instrumentation, acute lung injury was induced by infusion of OA (0.06 ml/kg; O1008, Sigma‐Aldrich, Castle Hill, NSW, Australia), with or without, intratracheal E. coli LPS (100 µg; O55:B5, Sigma‐Aldrich, Castle Hill, NSW, Australia) or intravenous E. coli LPS (1 μg/kg infused over 1 h; O55:B5, Sigma‐Aldrich, Castle Hill, NSW, Australia). Once a PaO₂/FiO₂ ratio <100 mmHg (PEEP ≥5 cmH₂O) was achieved (0 h), animals received protocolized intensive care for the duration of the study. At 6 h, animals were euthanized.