1 14c oleic acid

Vented T-25 tissue culture flasks were used to culture Mtb in 7H9 containing fatty acid-free albumin-dextrose supplemented with 0.01% glycerol and 0.05% tyloxapol. Once the bacteria reached the mid-logarithmic growth phase, cells were concentrated in a spent medium to a final OD₆₀₀ of 0.7. The bacterial cultures were immediately supplied 1.0 µCi of [1-¹⁴C]-oleic acid (Perkin Elmer) and were incubated at 37 °C. At the 5, 30, 60, and 120 min time points 1.5 ml of the bacterial samples were removed. Each sample was washed three times with cold PBS containing 0.1% Triton X-100 and 0.1% fatty acid-free BSA to remove surface-bound radiolabel. Following the three washes the bacteria were fixed in 4% paraformaldehyde (PFA) and ¹⁴C incorporation was quantified via scintillation counting. Radioactive counts at each time point were plotted and used for linear regression calculations to determine the rate of lipid uptake. The rates were normalized to wild type and expressed as uptake efficiency (%).

Cardiac myocytes were prepared from the hearts of control and PVR pups as described [16 (link)]. The protocol allows for rat cardiomyocytes to be cultured for up to 72 h at 37°C in 5% CO₂ without significant change in phenotype [17 (link)]. Enough hearts were collected to yield approximately 15-20million cardiac myocytes sufficient to plate 15–20 x 35mm dishes (1 million/dish, Corning Primeria^TM). Isolated cardiac myocytes were incubated with 0.1 mM [1,3-³H]glycerol (2 μCi/dish, Perkin Elmer) or 0.1 μM [1-¹⁴C]linoleic acid (2 μCi/dish, Perkin Elmer) bound to albumin (1:1 molar ratio) or 0.1 μM [1-¹⁴C]oleic acid (2 μCi/dish Perkin Elmer) bound to albumin (1:1 molar ratio) for up to 360 min (6 h) and radioactivity incorporated into phospholipids determined as previously described [18 (link)].

Fatty acid metabolism was assessed in intact (tendon-to-tendon isolated) EDL muscle examined 14 days after administration of AAV6:lacZ-shRNA or AAV6:Yap-shRNA^{71 (link)}. Muscle was incubated for 2 h in warmed (37 °C), pre-gassed (95% O₂–5% CO₂) low-glucose DMEM containing 2% fatty acid-free BSA, 500 µmol/L oleic acid and 1 µCi/mL ^1–14C-oleic acid (Perkin Elmer, Australia). Media was acidified in 1 mol/L perchloric acid, CO₂ captured in 1 mol/L NaOH, and radioactivity counted using a liquid-scintillation counter (TRI-CARB 4910TR 110 V Liquid-Scintillation Counter, Perkin Elmer). Tissue was homogenised in 2:1 (v/v) chloroform:methanol, phase separation initiated by addition of 0.9% NaCl, the aqueous layer used for assessment of acid-soluble metabolites and the lipid layer for assessment of TAG incorporation.

All dinapinones were purified from a culture broth of the microorganism T. pinophilus FKI-3864 according to established methods^{19 (link),21 (link)}. Vioxanthin was kindly gifted from Prof. Michael Muller at Institut für Biotechnologie 2, Jülich, Germany. [1-¹⁴C]oleic acid (1.85 GBq/mmol) and [¹⁴C]oleoyl-CoA (1.85 GBq/mmol) were purchased from PerkinElmer (Waltham, MA, U.S.A.). [1-¹⁴C]palmitoyl-CoA was purchased from Moravek Biochemicals (Brea, CA, U.S.A.). Phosphatidic acid, L-a-dioleoyl-[2-oleoyl-1-¹⁴C] (2.04 GBq/mmol) and glycerol, L-[¹⁴C(U)] 3-phosphate (5.55 GBq/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France). Penicillin (10,000 units/ml) and streptomycin (10,000 mg/ml) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Ham’s F-12 medium, G3P, BSA, 1,2-dioleoyl-sn-glycerol, palmitoyl-CoA, Triton X-100, poly L-lysine, oil red O, bafilomycin A₁, 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), isoproterenol and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). DMEM (high glucose) medium, diisopropyl fluorophosphates, 2-mercaptoethanol, oleic acid and hematoxylin were purchased from Wako (Osaka, Japan). Nonidet P-40 was purchased from Nacalai Tesque (Kyoto, Japan).