The main source of docosahexaenoic acid (DHA) used in the study was acquired from Sigma Aldrich, St. Louis, MO (D2534–100MG). Three additional sources of DHA were tested in the WNT reporter assays (D2534–1G, Sigma Aldrich; U-84-A, Nu-Chek Prep Inc., Elysian, MN; Cayman chemical, Ann Arbor, MI, 90310). Other compounds used in the study include: Oleic acid (Nu-Chek Prep Inc.; U-46-A), crebinostat (custom synthesized compound), CHIR-99021 (custom synthesis), forskolin (Sigma Aldrich; F6886), S-(5’-Adenosyl)-L-methionine chloride dihydrochloride (SAMe; Sigma Aldrich; A7007).
Analytical LC/MS was performed on a Waters 2545 HPLC equipped with a 2998 diode array detector, a 2424 evaporative light scattering detector, a 2475 multichannel fluorescence detector, and a Waters 3100 ESI-MS module, using a XTerraMS C18 5 μm, 4.6 × 50 mm column at a flow rate of 5 mL/min with a linear gradient (95% A: 5% B to 100% B 570 sec and 30 sec hold at 100% B, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid). LC/MS analyses were performed on DHA sources 1–4 (Supplemental Figure S7 ). For each DHA source, in addition to the dominant peak for DHA (m/z (M-H)- 327.3, [calculated C22H32O2: 328.24]), several minor components (less than 1%) conforming to various oxidized forms of DHA were detected.
Analytical LC/MS was performed on a Waters 2545 HPLC equipped with a 2998 diode array detector, a 2424 evaporative light scattering detector, a 2475 multichannel fluorescence detector, and a Waters 3100 ESI-MS module, using a XTerraMS C18 5 μm, 4.6 × 50 mm column at a flow rate of 5 mL/min with a linear gradient (95% A: 5% B to 100% B 570 sec and 30 sec hold at 100% B, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid). LC/MS analyses were performed on DHA sources 1–4 (