.gFigure 6. BEP-CE induces macropinocytosis and lipid accumulation in macrophages. A and B, J774 cells have been incubated with Alexa Fluor 488-labeled dextran (10,000 Da), inside the presence of 10 mg/ml AA-CE or BEP-CE, for 30 min. Cells were stained for F-actin (red) and nuclei (blue) and imaged (A, scale bar, ten mm) or analyzed by flow cytometry to quantify the amount of cells that internalized fluorescent dextran (B). C , J774 cells had been incubated with 200 mg/ml native LDL (protected from oxidation with BHT), within the presence of ten mg/ml AA-CE or BEP-CE, for 40 hours and stained for neutral lipid with LipidTox (red) and nuclei (blue) (C), or with Oil Red O and counterstained with hematoxilin (E). Scale bar, 20 mm. Lipid deposits have been quantified by measuring the location of LipidTox staining per cell (D). A total of 73 and 111 cells in AA-CE and BEP-CE samples, respectively, have been measured in three independent experiments.2832911-62-1 In stock Mean6SE. *, p,0.05. doi:10.1371/journal.pone.0083145.gPLOS One | plosone.orgOxidized Cholesterol Ester Activates TLRFigure 7. BEP-CE in human plasma and atherosclerotic lesions. LC-MS/MS spectra obtained using the MRM system detecting the 754.9 precursor and 369.4 item masses. A, Solution of an AA-CE/15LO reaction. B, Lipid extracts from plasma samples obtained from patients presented for cardiac catheterization. Shown are 2 out of 12 samples tested, corresponding to # three (solid line) and #8 (dashed line) in panel D. C, Lipid extracts from the embolic material captured in distal protection devices during saphenous vein graft (solid line; #1 in E) and carotid artery (dashed line; #8 in E) interventions. Samples in panels B and C had been collected from different individuals. Shaded will be the peaks widespread for all 3 groups of samples. D, Relative intensity (arbitrary units) on the m/z = 755, eight.28 min retention time signal in 12 human plasma samples tested. E, Relative intensity (arbitrary units) in the m/z = 755, eight.27 min retention time signal in human plaque samples. SVG, saphenous vein graft; SFA, superficial femoral artery; Renal, renal artery; Carotid, carotid artery. doi:10.1371/journal.pone.0083145.gwe then utilised OxCE derived from a reaction of AA-CE with 15LO. The item of this reaction induced biological responses in macrophages similar to those induced by mmLDL [14]. We as a result oxidized AA-CE within a 15LO enzymatic reaction and separated the resulting mixture of OxCE items by the LC technique described in Strategies.Exatecan Intermediate 2 Formula The LC fractions have been tested with J774 macrophages to assay their effects on cell spreading and phosphorylation of ERK1/2, the two robust effects of nonfractionated AA-CE/15LO and mmLDL [14].PMID:23439434 As shown in Fig. 1, fraction #17 induced probably the most visible cell spreading and ERK1/2 phosphorylation. The dominant mass in #17 OxCE fraction had an m/z = 755 (ammonium adduct; exact mass 754.9 precursor and 369.4 product). Depending on a study from Porter’s group, who identified a lot of products of free of charge radical oxidation of AA-CE and of 15(S)-HETE [33], the m/z = 755 mass most likely represents an oxidized AACE with each bicyclic endoperoxide and hydroperoxide groups, right here abbreviated as BEP-CE. Next, using the Porter group’smethodology, we reproduced their AMVN-initiated free of charge radical oxidation of AA-CE and their separation protocol [33] to isolate the m/z = 755 compound and subjected it to Ag+ CIS-MS/MS as they described [34]. The fragmentation pattern of an m/z = 755 AA-CE/AMVN solution was consistent with all the structure of cholestery.