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• W2110135444 abstract "Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. ERRATAJournal of Lipid ResearchVol. 54Issue 2PreviewThe Journal has been informed that Figure 2 in the print and online versions of “Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes” (J. Lipid Res. 2012. 53: 2343–2354) was incorrect. The online version has been corrected and the correct figure appears below. Full-Text PDF Open Access Lipid droplets (LD) are cytosolic inclusions present in most eukaryotic cells that contain a core rich in neutral lipids such as triacylglycerol (TAG) and cholesteryl esters (CE) and are surrounded by a phospholipid monolayer decorated with a variety of proteins, such as PAT family proteins (perilipin, adipose differentiation related protein, and tail-interacting protein of 47 kDa [TIP-47]) and caveolins (1Tauchi-Sato K. Ozeki S. Houjou T. Taguchi R. Fujimoto T. The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition.J. Biol. Chem. 2002; 277: 44507-44512Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar–2Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (760) Google Scholar, 3Thiele C. Spandl J. Cell biology of lipid droplets.Curr. Opin. 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Macrophage cholesteryl ester mobilization and atherosclerosis.Vascul. Pharmacol. 2010; 52: 1-10Crossref PubMed Scopus (108) Google Scholar–8Greenberg A.S. Coleman R.A. Kraemer F.B. McManaman J.L. Obin M.S. Puri V. Yan Q.W. Miyoshi H. Mashek D.G. The role of lipid droplets in metabolic disease in rodents and humans.J. Clin. Invest. 2011; 121: 2102-2110Crossref PubMed Scopus (454) Google Scholar, 9Olofsson S.O. Andersson L. Haversen L. Olsson C. Myhre S. Rutberg M. Mobini R. Li L. Lu E. Boren J. et al.The formation of lipid droplets: possible role in the development of insulin resistance/type 2 diabetes.Prostag. Leukot. Essent. Fatty Acids. 2011; 85: 215-218Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 10Bozza P.T. Magalhaes K.G. Weller P.F. Leukocyte lipid bodies. Biogenesis and functions in inflammation.Biochim. Biophys. Acta. 2009; 1791: 540-551Crossref PubMed Scopus (182) Google Scholar). LDs are generated by cells under different environmental conditions, suggesting a distinct pathophysiological significance for each of these conditions. Cells generate lipid droplets from exogenous lipid sources, especially free fatty acids and cholesterol from serum lipoproteins (11Gubern A. Casas J. Barcelo-Torns M. Barneda D. de la Rosa X. Masgrau R. Picatoste F. Balsinde J. Balboa M.A. Claro E. Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets.J. Biol. Chem. 2008; 283: 27369-27382Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar–12Gubern A. Barceló-Torns M. Barneda D. Lopez J.M. Masgrau R. Picatoste F. Chalfant C.E. Balsinde J. Balboa M.A. Claro E. JNK and ceramide kinase govern the biogenesis of lipid droplets through activation of group IVA phospholipase A2.J. Biol. Chem. 2009; 284: 32359-32369Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13den Hartigh L.J. Connolly-Rohrbach J.E. Fore S. Huser T.R. Rutledge J.C. 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Lipid Res. 2007; 48: 1280-1292Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), probably with an energy-storage purpose; however, when cells are under different stress signals, LD biogenesis occurs in the absence of external lipid via rearrangement of membrane phospholipids and fatty acids into newly formed TAG molecules (16Gubern A. Barceló-Torns M. Casas J. Barneda D. Masgrau R. Picatoste F. Balsinde J. Balboa M.A. Claro E. Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2.J. Biol. Chem. 2009; 284: 5697-5708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Iorio E. Di Vito M. Spadaro F. Ramoni C. Lococo E. Carnevale R. Lenti L. Strom R. Podo F. Triacsin C inhibits the formation of 1H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells.Biochim. Biophys. Acta. 2003; 1634: 1-14Crossref PubMed Scopus (49) Google Scholar). The leukocytes, cells typically associated with inflammatory reactions can induce the rapid formation of LDs when exposed to proinflammatory stimuli (18Weller P.F. Ackerman S.J. Nicholson-Weller A. Dvorak A.M. Cytoplasmic lipid bodies of human neutrophilic leukocytes.Am. J. Pathol. 1989; 135: 947-959PubMed Google Scholar–19Weller P.F. Ryeom S.W. Picard S.T. Ackerman S.J. Dvorak A.M. Cytoplasmic lipid bodies of neutrophils: formation induced by cis-unsaturated fatty acids and mediated by protein kinase C.J. Cell Biol. 1991; 113: 137-146Crossref PubMed Scopus (80) Google Scholar, 20Pacheco P. Bozza F.A. Gomes R.N. Bozza M. Weller P.F. Castro-Faria-Neto H.C. Bozza P.T. Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular loci involved in eicosanoid metabolism.J. Immunol. 2002; 169: 6498-6506Crossref PubMed Scopus (115) Google Scholar, 21Bozza P.T. Viola J.P. Lipid droplets in inflammation and cancer.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 243-250Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Moreover, it is becoming increasingly recognized that LDs are specialized intracellular sites for the biosynthesis and amplification of the eicosanoid biosynthetic response during inflammation (10Bozza P.T. Magalhaes K.G. Weller P.F. Leukocyte lipid bodies. Biogenesis and functions in inflammation.Biochim. Biophys. Acta. 2009; 1791: 540-551Crossref PubMed Scopus (182) Google Scholar, 20Pacheco P. Bozza F.A. Gomes R.N. Bozza M. Weller P.F. Castro-Faria-Neto H.C. Bozza P.T. Lipopolysaccharide-induced leukocyte lipid body formation in vivo: innate immunity elicited intracellular loci involved in eicosanoid metabolism.J. Immunol. 2002; 169: 6498-6506Crossref PubMed Scopus (115) Google Scholar–21Bozza P.T. Viola J.P. Lipid droplets in inflammation and cancer.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 243-250Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 22Bozza P.T. Bakker-Abreu I. Navarro-Xavier R.A. Bandeira-Melo C. Lipid body function in eicosanoid synthesis: an update.Prostaglandins Leukot. Essent. Fatty Acids. 2011; 85: 205-213Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Decided amounts of arachidonic acid (AA) are present in the phospholipid monolayer surrounding the LD, and a variety of enzymes involved in AA metabolism have been demonstrated to localize in LDs (23Accioly M.T. Pacheco P. Maya-Monteiro C.M. Carrossini N. Robbs B.K. Oliveira S.S. Kaufmann C. Morgado-Diaz J.A. Bozza P.T. Viola J.P. Lipid bodies are reservoirs of cyclooxygenase-2 and sites of prostaglandin E2 synthesis in colon cancer cells.Cancer Res. 2008; 68: 1732-1740Crossref PubMed Scopus (244) Google Scholar–24Wooten R.E. Willingham M.C. Daniel L.W. Leslie C.C. Rogers L.C. Sergeant S. O'Flaherty J.T. Novel translocation responses of cytosolic phospholipase A2α fluorescent proteins.Biochim. Biophys. Acta. 2008; 1783: 1544-1550Crossref PubMed Scopus (26) Google Scholar, 25Moreira L.S. Piva B. Gentile L.B. Mesquita-Santos F.P. D'Avila H. Maya-Monteiro C.M. Bozza P.T. Bandeira-Melo C. Diaz B.L. Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells.Biochim. Biophys. Acta. 2009; 1791: 156-165Crossref PubMed Scopus (45) Google Scholar, 26Valdearcos M. Esquinas E. Meana C. Gil-de-Gómez L. Guijas C. Balsinde J. Balboa M.A. Subcellular localization and cellular role of lipin-1 in human macrophages.J. Immunol. 2011; 186: 6004-6013Crossref PubMed Scopus (60) Google Scholar). One of these enzymes is the group IVA phospholipase A2, also known as cytosolic phospholipase A2α (cPLA2α), a central enzyme for the release of AA from phospholipids (27Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Regulation and inhibition of phospholipase A2.Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar–28Balsinde J. Winstead M.V. Dennis E.A. Phospholipase A2 regulation of arachidonic acid mobilization.FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar, 29Leslie C.C. Regulation of arachidonic acid availability for eicosanoid production.Biochem. Cell Biol. 2004; 82: 1-17Crossref PubMed Scopus (107) Google Scholar, 30Pérez-Chacón G. Astudillo A.M. Balgoma D. Balboa M.A. Balsinde J. Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases.Biochim. Biophys. Acta. 2009; 1791: 1103-1113Crossref PubMed Scopus (138) Google Scholar, 31Shimizu T. Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 123-150Crossref PubMed Scopus (406) Google Scholar). cPLA2α is phosphorylated and activated by members of the mitogen-activated protein kinase family of enzymes (i.e., the extracellular-regulated kinases [ERK] p42/p44, p38, and c-Jun N-terminal kinase [JNK]), although the specific form involved appears to depend on cell type and stimulus (30Pérez-Chacón G. Astudillo A.M. Balgoma D. Balboa M.A. Balsinde J. Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases.Biochim. Biophys. Acta. 2009; 1791: 1103-1113Crossref PubMed Scopus (138) Google Scholar). In this work we have examined the pathways for LD biosynthesis in human monocytes exposed to free AA and have identified the signaling cascade and intracellular events leading to LD formation in human monocytes. On one hand, AA may just serve as a lipid source for TAG biosynthesis and subsequent LD formation; on the other hand, AA concomitantly activates the MAP kinases p38 and JNK, both of which promote LD formation in a manner that depends on a biologically active cPLA2α enzyme. Cell culture medium and BODIPY® 493/503 were obtained from Molecular Probes-Invitrogen (Carlsbad, CA). Chloroform and methanol (HPLC grade) were from Fisher Scientific (Hampton, NH). [5,6,8,9,11,12,14,15-3H]AA (sp. act. 211 Ci/mmol) was purchased from GE Healthcare (Buckinghamshire, UK). [1,2-14C]acetic acid (sp. act. 54.3 mCi/mmol) was from Perkin Elmer (Waltham, MA). Silicagel thin layer chromatography plates were from Macherey-Nagel (Düren, Germany). The p38 MAP kinase inhibitor SB 203580 was from Calbiochem/Merck KGaA (Darmstadt, Germany). Triacsin C was purchased from Enzo Life Sciences (Farmingdale, NY). Paraformaldehyde was from Electron Microscopy Sciences (Hartfield, PA). Antibodies against p-cPLA2(Ser505), p-p38(Thr180/Tyr182) and p-JNK(Thr183/Tyr185) were purchased from Cell Signaling (Danvers, MA). The cPLA2α inhibitor pyrrophenone was synthesized and generously provided by Dr. Amadeu Llebaria (Institute for Chemical and Environmental Research, Barcelona, Spain) (32Pérez R. Matabosch X. Llebaria A. Balboa M.A. Balsinde J. Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells.J. Lipid Res. 2006; 47: 484-491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). All other reagents were from Sigma-Aldrich. Human monocytes were isolated from buffy coats of healthy volunteer donors obtained from the Centro de Hemoterapia y Hemodonación de Castilla y León (Valladolid, Spain). Written informed consent was obtained from each donor. Briefly, blood cells were diluted 1:1 with PBS, layered over a cushion of Ficoll-Paque, and centrifuged at 750 g for 30 min. The mononuclear cellular layer was recovered and washed three times with PBS, resuspended in RPMI 1640 medium supplemented with 40 μg/ml gentamicin, and allowed to adhere in sterile dishes for 2 h at 37°C in a humidified atmosphere of CO2/air (1:19). Nonadherent cells were removed by washing extensively with PBS, and the remaining attached monocytes were used the following day. Human macrophages were obtained by incubating plastic-adhered monocytes in RPMI with heat-inactivated 5% human serum for 2 weeks in the absence of exogenous cytokine mixtures. Fatty acids were dissolved in ethanol, and an appropriate aliquot was diluted in the incubation medium to obtain the desired concentration. When radioactive fatty acids were used, they were spiked into an ethanol solution containing cold fatty acids to generate the required specific radioactivity before adding them to the incubation media. Ethanol concentrations in the incubation media were always below 0.1%, and the appropriate controls were run to ensure that ethanol had no effect on its own on cells. When inhibitors were used, they were added to the incubation media 30 min before treating the cells with AA. For all experiments, the cells were incubated in media consisting of serum-free RPMI 1640 medium (supplemented with 2 mM L-glutamine) at 37°C in a humidified 5% CO2 atmosphere. After incubations, the cells were washed twice with PBS, and a cell extract corresponding to 107 cells was scraped in ice-cold water and sonicated in a tip homogenizer twice for 15 s. Before extraction and separation of lipid classes, internal standards were added. For total phospholipids, 10 nmol of 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine was added; for TAG, 10 nmol of 1,2,3-triheptadecanoylglycerol was added; and for CE, 30 nmol of cholesteryl tridecanoate was added. Total lipids were extracted according to Bligh and Dyer [33], and the resulting lipid extract was separated by thin-layer chromatography using n-hexane/diethyl ether/acetic acid (70:30:1, by vol.) as the mobile phase. Spots corresponding to the various lipid classes were scraped, and phospholipids were extracted from the silica with 800 µl methanol followed by 800 µl chloroform/methanol (1:2, v/v) and 500 µl chloroform/methanol (2:1, v/v). TAG and CE were extracted with 1 ml chloroform/methanol (1:1, v/v) followed by 1 ml of chloroform/methanol (2:1, v/v). Glycerolipids were transmethylated with 500 μl of 0.5 M KOH in methanol for 30 min at 37°C. To neutralize, 500 μl of 0.5 M HCl was added. Cholesteryl esters were transmethylated as follows. Each fraction was resuspended in 400 μl of methyl propionate, and 600 μl of 0.84 M KOH in methanol was added for 1 h at 37°C. Afterward, 50 μl and 1 ml of acetic acid and water, respectively, were added to neutralize. Extraction of fatty acid methyl esters was carried out with 1 ml n-hexane twice. Analysis of fatty acid methyl esters was carried out in a Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (70 eV) equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length × 250 µm internal diameter × 0.15 µm film thickness) under the conditions described previously (34Astudillo A.M. Perez-Chacón G. Balgoma D. Gil-de-Gómez L. Ruipérez V. Guijas C. Balboa M.A. Balsinde J. Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells.Biochim. Biophys. Acta. 2011; 1811: 97-103Crossref PubMed Scopus (38) Google Scholar, 35Astudillo A.M. Pérez-Chacón G. Meana C. Balgoma D. Pol A. del Pozo M.A. Balboa M.A. Balsinde J. Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis.J. Biol. Chem. 2011; 286: 35299-35307Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with a slight modification of the procedure to improve separation of fatty acid methyl esters. Briefly, oven temperature was held at 50°C for 1 min, increased to 175°C at a rate of 25°C/min, increased to 215°C at a rate of 1.5°C/min, with the final ramp being reached at 235°C at a rate of 10°C/min. The final temperature was maintained for 5 min, and the run time was 39.67 min. Data analysis was carried out with the Agilent G1701EA MSD Productivity Chemstation software, revision E.02.00. Monocytes preincubated with various concentrations of triacsin C were exposed to 3 nM [3H]AA (0.25 μCi/ml) or 7 nM [3H]palmitic acid (0.25 µCi/ml) for 30 min. Afterward, the cells were washed four times with PBS containing 0.5% albumin to remove the fatty acid that had not been incorporated. Cells were scraped twice with 0.1% Triton X-100 in PBS, and total lipids were extracted according to the method of Bligh and Dyer (33Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar), reconstituted in chloroform/methanol (2:1, v/v), and separated by thin-layer chromatography with n-hexane/ether/acetic acid (70:30:1, v/v/v). The spots corresponding to TAG were cut out, and the plate and analyzed for radioactivity by liquid scintillation counting (36Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.Proc. Natl. Acad. Sci. USA. 1995; 92: 8527-8531Crossref PubMed Scopus (256) Google Scholar–37Balsinde J. Dennis E.A. The incorporation of arachidonic acid into triacylglycerol in P388D1 macrophage-like cells.Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar, 38Pérez-Chacón G. Astudillo A.M. Ruipérez V. Balboa M.A. Balsinde J. Signaling role for lysophosphatidylcholine acyltransferase 3 in receptor-regulated arachidonic acid reacylation reactions in human monocytes.J. Immunol. 2010; 184: 1071-1078Crossref PubMed Scopus (50) Google Scholar). For these experiments, [14C]acetic acid (0.1 µCi/ml) was added to the cells at the time they were treated or not with 10 µM AA plus 3 µM triacsin C for 2 h. Afterward, the reactions were stopped, and the cell monolayers were scraped twice with 0.1% Triton X-100 in PBS. Lipids were extracted according to the method of Bligh and Dyer (33Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar). The total lipid fraction was subjected to alkaline hydrolysis and, after re-extraction, total 14C-radioactivity levels in the organic phase were determined by scintillation counting. For viability assays, monocytes were cultured in 96-well microtiter plates. At the end of the different treatments, viability was measured by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Biotech Iberica, Madrid, Spain). This is a colorimetric method for determining the number of viable cells in culture. The solution contains a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) that, when reduced, produces a colored formazan product that absorbs maximally at 490 nm. The compound is reduced due to the mitochondrial activity in the cell. The amount of formazan that is produced is directly proportionally to the number of viable cells. RNA was extracted using the TRIzol reagent method (Invitrogen) according to the manufacturer's protocol. First-strand cDNA was obtained by using the Moloney murine leukemia virus reverse transcriptase from 1 µg of RNA. PCR was then performed using specific primers for long-chain acyl-CoA synthetases as follows: 5′-ccagaagggcttcaagactg-3′ (forward) and 5′-gccttctctggcttgtcaac-3′ (reverse) for ACSL-1, 5′-catcgccatcttctgtgaga-3′ (forward) and 5′-ggtggctttccatcaacagt-3′ (reverse) for ACSL-3, 5′-ccgacctaagggagtgatga-3′ (forward) and 5′-cctgcagccataggtaaagc-3′ (reverse) for ACSL-4, and 5′-accagtggctgtcctaccag-5′ (forward) and 5′-gctgatgtccgctgtattga-3′ (reverse) for ACSL-6. Cycling conditions were as follows: 1 cycle at 95°C for 10 min, 35 cycles at 95°C for 30 s, 58°C for 45 s, 72°C for 1 min, and a final extension at 72°C for 10 min. Quantitative PCR was carried out with an ABI 7500 machine (Applied Biosystems, Carlsbad, CA) using the Brilliant III Ultra-Fast SYBR®Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA) and specific primers for each gene as follows: acetyl-CoA carboxylase, 5′-tcacacctgaagaccttaaagcc-3′ (forward) and 5′-agcccacactgcttgtactg-3′ (reverse); fatty acid synthase, 5′-acagcggggaatgggtact-3′ (forward) and 5′-gactggtacaacgagcggat-3′ (reverse); stearoyl-CoA desaturae, 5′-ttcctacctgcaagttctacacc-3′ (forward) and 5′-ccgagctttgtaagagcggt-3′ (reverse); very long-chain fatty acid elongase-6, 5′-aacgagcaaagtttgaactgagg-3′ (forward) and 5′-tcgaagagcaccgaatatactga-3′ (reverse). Cycling conditions were: 1 cycle at 95°C for 3 min and 40 cycles at 95°C for 12 s, 60°C for 15 s and 72°C for 28 s. The relative mRNA abundance for a given gene was calculated using the algorithm 2−ΔΔCt, with β-actin and cyclophilin A as internal standards (39Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real time quantitative PCR and the 2(-ΔΔC(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar). For these experiments, the cells were plated on coverslips on the bottom of 6-well dishes in a volume of 2 ml. The cells were fixed with 1 ml of 4% paraformaldehyde in PBS containing 3% sucrose for 20 min. Afterward, paraformaldehyde was removed by washing the cells thrice with PBS, and BODIPY493/503 and DAPI stainings were carried out by treating cells with these dyes at concentrations of 2 μg/ml and 1 μg/ml, respectively, in PBS for 10 min. Coverslips were mounted on microscopy slides with 25 μl of a polyvinyl alcohol solution until analysis by fluorescence microscopy. Fluorescence was monitored by microscopy using a NIKON Eclipse 90i device equipped with a CCD camera (model DS-Ri1; Nikon, Tokyo, Japan). A mercury HBO excitation lamp (Osram, Munich, Germany) was used, and the fluorescence from DAPI and BODIPY493/503 was recovered using the combination of a UV-2A (Ex 330-380; DM 400; BA 420) and a B-2A (Ex 450-490; DM 505; BA 520) filter, respectively. Images were analyzed with the software NIS – Elements (Nikon). Green and blue channels were merged with the Image-J software (http://rsb.info.nih.gov/ij/). Ca2+-dependent PLA2 activity was measured using a modification of the mammalian membrane assay described by Diez et al. [40Diez E. Chilton F.H. Stroup G. Mayer R.J. Winkler J.D. Fonteh A.N. Fatty-acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate.Biochem. J. 1994; 301: 721-726Crossref PubMed Scopus (91) Google Scholar]. Briefly, monocyte homogenates were incubated for 1–2 h at 37°C in 100 mM HEPES (pH 7.5) containing 1.3 mM CaCl2 and 100,000 dpm [3H]AA-labeled membrane, used as a substrate, in a final volume of 0.15 ml. Before assay, the cell membrane substrate was heated at 57°C for 5 min to inactivate CoA-independent transacylase activity (41Winkler J.D. Sung C.M. Bennett C.F. Chilton F.H. Characterization of CoA-independent transacylase activity in U937 cells.Biochim. Biophys. Acta. 1991; 1081: 339-346Crossref PubMed Scopus (44) Google Scholar). The assay contained 25 μM bromoenol lactone to completely inhibit endogenous Ca2+-independent PLA2 activity [36]. After lipid extraction, free [3H]AA was separated by thin-layer chromatography, using n-hexane/ethyl ether/acetic acid (70:30:1) as a mobile phase. For Ca2+-independent PLA2 activity, the cell homogenates were incubated for 2 h at 37°C in 100 mM HEPES (pH 7.5) containing 5 mM EDTA and 100 mM labeled phospholipid substrate (1-palmitoyl-2-[3H]palmitoyl-glycero-3-phosphocholine, sp. act. 60 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) in a final volume of 150 ml. The phospholipid substrate was used in the form of sonicated vesicles in buffer. The reactions were quenched by adding 3.75 volumes of chloroform/methanol (1:2). After lipid extraction, free [3H]palmitic acid was separated by thin-layer chromatography, using n-hexane/ ethyl ether/acetic acid (70:30:1) as a mobile phase. In some experiments, Ca2+-independent PLA2 activity was also measured using a mixed-micelle substrate or the natural membrane assay. For the mixed micelle assay, Triton X-100 was added to the dried lipid substrate at a molar ratio of 4:1. Buffer was added, and the mixed micelles were made by a combination of heating above 40°C, vortexing, and water bath sonication until the solution clarified. The natural membrane assay was carried out exactly as described above, except that CaCl2 was omitted and 5 mM EDTA was added instead. All of these assay conditions have been validated previously regarding time, homogenate protein, and substrate concentration (42Balsinde J. Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling.Biochem. J. 2002; 364: 695-702Crossref PubMed Scopus (74) Google Scholar–43Balboa M.A. Balsinde J. Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells.J. Biol. Chem. 2002; 277: 40384-40389Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 44Balboa M.A. Sáez Y. Balsinde J. Calcium-independent phospho" @default.
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• W2110135444 title "Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes" @default.
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