13 s hode

Vasicine, vasicinone, isoorientin, isovitexin, vitexin, apigenin, 12,13-DiHOME, 9,10-DiHOME, 13(S)-HODE, 9(S)-HODE, 13-oxoODE, 9-oxoODE, 12,13-EpOME and 9,10-EpOME, 9-HpODE, and 7-ethoxyresorufin were purchased from Cayman Chemical (Ann Arbor, MI, USA). AITC, ionomycin calcium salt, HC-030039, and A967079 were from Sigma-Aldrich (St. Louis, MO, USA). DMSO was from Fisher Scientific (Waltham, MA, USA).

Binding affinities of PPARγ LBD WT and mutants (R280C, C285Y, Q286E, Q286P, F287Y, R288C, R288H and S289C) for 15-deoxy-∆^12,14-prostaglandin J₂ (15d-PGJ2, Sigma Aldrich) and 13S-hydroxy-9Z,11E-octadecadienoic acid (13S-HODE, Cayman Chemical, Ann Arbor, MI, USA) were measured using a MicroCal ITC200 system (Malvern Instruments, Worcestershire, UK). All the measurements were conducted in the buffer of PBS pH 7.2 at 25 °C. The ligands filled in the syringe were titrated into the proteins in the cell using 19 injections of 2 μL and an initial injection of 0.4 μL with 150 s equilibration between injections. The final protein concentrations of PPARγ LBD WT and mutants were 60 μM for 15d-PGJ2 at the ligand concentration of 900 μM and 45 μM for 13S-HODE at the ligand concentration of 900 μM, respectively. The obtained data were analyzed using Origin 7.0 software.

OTA standard (purity ≥ 99%) was purchased from Fermentek (Jerusalem, Israel). cAMP standard (purity ≥ 98%) was purchased from Solar Bio (Beijing China). Oxylipins 9(S)-HODE (purity ≥ 98%), 13(S)-HODE (purity ≥ 98%), and PGE₂ (purity ≥ 98%) were purchased from Cayman Chemical (Michigan, USA). The stock solutions were dissolved in ethanol and stored at − 80 °C, and then diluted with ethanol to working solutions before analysis. Other conventional solvents used in this experiment are chromatographic purity, all purchased from Merck (Darmstadt, Germany).

When (SR−) MCF-7 human breast cancer xenografts reached 5–6 gm estimated tumor weight, animals were prepared for arterial-venous difference measurements of total fatty acids (TFAs), LA, 13-HODE, glucose, lactate, pO₂, CO₂ and pH between 0600 and 1000 hrs when endogenous melatonin levels were low. Tissue-isolated xenografts were perfused in situ with rat donor whole-blood to which either synthetic melatonin (Sigma, St. Louis, MO) and/or 13(S)-HODE (Cayman Chemicals, Ann Arbor, MI), or MT₁/MT₂ melatonin receptor antagonist S20928 (a generous gift from Institute de Recherches Internationales Servier, Courbevoie Cedex, France) was added as previously described [9] (link), [10] (link), [12] (link), [28] (link). Sets (3 or 4 tumors/perfusion) of tissue-isolated human breast cancer xenografts were perfused in situ for 1 hr as previously described [9] (link), [10] (link), [12] (link), [28] (link) with whole-blood collected from donor rats. Incorporation of [³H]thymidine into tumor DNA was initiated 20 minutes prior to the end of each perfusion by injecting 20 µl of physiological saline containing 2 µCi [methyl-³H]thymidine/gm (New England Nuclear, Perkin Elmer, Boston, MA) estimated tumor weight into the arterial catheter. At the completion of each perfusion, tumors were freeze-clamped under liquid nitrogen, weighed and store at −85°C until analysis.