Linolenic acid

Oleic acid (94%), linOleic acid (99%), linolenic acid (68%), racemic γ-dodecalactone, acetic anhydride, pyridine, glucose, peptone, casein peptone, yeast extract, and tryptic soy broth were purchased from Sigma-Aldrich (Milan, Italy). Trimethylsilyldiazomethane 10% solution in hexane (TCI Europe N.V.) was purchased from Zentek srl (Milan, Italy).
Optically enriched (R)-10-hydroxystearic acid was prepared by Lactobacillus rhamnosus-mediated hydration of Oleic acid, according to the biotransformation procedure described in our previous work [33 (link)]. A reference standard sample of 10-ketostearic acid was prepared by oxidation of (R)-10-hydroxystearic acid using the Jones reagent [33 (link)]. A reference standard of 4-ketolauric acid was prepared by transformation of γ-dodecalactone into ethyl 4-hydroxylaurate followed by chromic oxidation, according to the procedure reported by Horikawa et al. [72 (link)]. All the compounds were previously isolated and their structures were confirmed by NMR analyses [30 (link),33 (link)].

Colonies of HS401 cells were collected by incubation with 1 mg/ml Collagenase IV (Worthington Biochemical Corporation) for 40 min in 37°C and plated as small clumps on SNL feeders in 1:3 ratio using mTeSR1 medium. The next day, medium was changed to SSC-differentiation medium [5 (link)] consisting of basal medium α-MEM (Life Technologies), 0.2% w/v bovine serum albumin (BSA, Sigma), 1x Glutamax (Life Technologies), 10 mM HEPES (Sigma), 50 U/ml and 50 mg/ml penicillin-streptomycin, 50 μM β-mercaptoethanol (Life Technologies), 5 μg/ml human recombinant insulin (Sigma), 10 μg/ml holo-transferrin (Sigma), 30 nM sodium selenite (Sigma), 60 μM putrescine (Sigma), 2.36 μM palmitic acid (Sigma), 0.21 μM palmitoleic acid (Sigma), 0.88 μM stearic acid (Sigma), 1.02 μM oleic acid (Sigma), 2.71 μM linoleic acid (Sigma), 0.43 μM linolenic acid (Sigma), 1 ng/ml human recombinant bFGF (R&D Systems) and 20 ng/ml recombinant human GDNF (R&D Systems). The SSC-differentiation medium was gassed with 90% N₂, 5% CO_2, 5% O₂ gas mixture for 30s before changed to cells every two days.

LOX activity was spectrophotometrically assayed by recording the increase in absorbance at 234 nm, using linolenic acid (C18:3) as substrate according to Surrey (1964), with some modification. Briefly, the assay was performed at 25 °C in a reaction medium (1 mL) containing 100 mM borate buffer pH 9.0 and 16 µM of linolenic acid (Sigma) prepared in 95% dimethyl sulfoxide (DMSO), and the reaction was initiated by the addition of 10 µL purified soybean LOX (dilution 1:4000). One unit of LOX activity is defined as the amount of enzyme that causes an increase in A₂₃₄ of 0.001 per min at pH 9.0 at 25 °C when linolenic acid is used as a substrate.
Protein concentration was determined using the Bio-Rad protein assay (Hercules, CA), with bovine serum albumin as standard. Band intensity was quantified using ImageJ 1.45 software.

C2C12 cells were plated at 10,000 cells/well in a 96-well plate and allowed to adhere overnight. The next day, medium was replaced with fresh DMEM containing either vehicle (isopropanol) or 20 μM CoQ additive (Sigma) in isopropanol. After 24 h, cells were washed with dPBS, and media were replaced with DMEM containing 10 mM galactose with varying concentrations of simvastatin (Sigma, S6196) in isopropanol and/or linolenic acid (Sigma, L2376) in isopropanol. After 48 h, media were removed, and cells were washed three times with dPBS. Crystal violet staining solution (0.5% crystal violet in 25% methanol) was added to the cells and allowed to incubate for 20 min with gentle rocking. After removing the staining solution, cells were washed three times with dPBS, and remaining crystal violet was dissolved with 100% methanol. Absorbance was read at 540 nm in a microplate reader (Cytation3).

Recombinant human BMP2 was purchased from R&D Systems (Minneapolis, MN). E2 was purchased from Steraloids, Inc. (Newport, RI). Estradiol 17β cypionate was purchased from Pfizer Company (Kalamazoo, MI). Phenol red-free DMEM, linolenic acid, bovine serum albumin (BSA), and fine chemicals were purchased from Sigma Chemical Company (St. Louis, MO). LDN193,189 was purchased from Sigma Chemical Co. (St. Louis, MO), ICI182,780 was purchased from Tocris Bioscience (Bristol, UK), and human holo-transferrin was obtained from BD pharmaceuticals (San Jose, CA). Plastic embedding medium and supplies were obtained from Electron Microscopy Sciences (Hatfield, Pa). Quantitative RT-PCR primers and probes were synthesized by Sigma Chemical Company. RNeasy mini kit and Taq DNA polymerase were from Qiagen, Inc. (Valencia, CA). All other molecular biology grade chemicals were obtained from Sigma Chemical Company or Fisher Scientific (Pittsburgh, PA).