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lta4 methyl ester  (Larodan)


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    Larodan lta4 methyl ester
    Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and <t>LTA4-H</t> in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).
    Lta4 Methyl Ester, supplied by Larodan, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lta4 methyl ester/product/Larodan
    Average 90 stars, based on 4 article reviews
    lta4 methyl ester - by Bioz Stars, 2026-02
    90/100 stars

    Images

    1) Product Images from "Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase."

    Article Title: Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

    Journal: Scientific reports

    doi: 10.1038/s41598-017-09795-w

    Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and LTA4-H in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).
    Figure Legend Snippet: Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and LTA4-H in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).

    Techniques Used: Activity Assay, Recombinant, Fluorescence, Cell-Free Assay, Control, Incubation, Tandem Mass Spectroscopy

    Figure 3. Diflapolin shows target specificity within the AA cascade without cytotoxicity. (a) mPGES-1 activity. Microsomes of IL-1β-stimulated A549 cells were pretreated with diflapolin, 10 µM MK886, or vehicle for 10 min on ice and stimulated with 20 µM PGH2. After 1 min at 4 °C, PGE2 formation was analyzed by HPLC. (b) LTC4S activity. Microsomes of LTC4S-expressing HEK293 cells were pretreated with diflapolin, MK886 (10 µM), or vehicle for 10 min on ice with subsequent addition of LTA4-methyl ester. After 10 min at 4 °C, LTC4-methyl ester was analyzed by UPLC-MS/MS. (c) COX-1/2 activity. In cell-free assays, purified ovine COX-1 and recombinant human COX-2 were pretreated with diflapolin (10 µM), indometacin (10 µM) or vehicle (0.1% DMSO) for 5 min on ice and stimulated with AA (5 and 2 µM for COX-1 and -2, respectively for 10 min at 37 °C. 12-HHT was analyzed by HPLC. (d) Effect of diflapolin on 12- and 15-LOX. Intact neutrophils were pre- incubated by diflapolin or vehicle (0.1% DMSO) and stimulated with 2.5 µM Ca2+-ionophore plus 20 µM AA for 10 min at 37 °C. 12- and 15-HETE were determined by HPLC. (e) AA release. [3H]AA-labeled neutrophils were pretreated with diflapolin (1 µM), RSC-3388 (10 µM) or vehicle (0.1% DMSO) and stimulated by 2.5 µM Ca2+- ionophore for 15 min. Radioactivity of the supernatant was analyzed by scintillation counting. (f) Cell viability assay. Monocytes were treated with diflapolin (10 µM), staurosporin (3 µM) or vehicle (0.3% DMSO) for 24 and 48 h, respectively. Cell viability was determined by MTT assay. Data are expressed as percentage of control (100%), means + SEM, n = 3–5. *p < 0.05; **p < 0.005; ***p < 0.001 vs. vehicle control (paired t-test).
    Figure Legend Snippet: Figure 3. Diflapolin shows target specificity within the AA cascade without cytotoxicity. (a) mPGES-1 activity. Microsomes of IL-1β-stimulated A549 cells were pretreated with diflapolin, 10 µM MK886, or vehicle for 10 min on ice and stimulated with 20 µM PGH2. After 1 min at 4 °C, PGE2 formation was analyzed by HPLC. (b) LTC4S activity. Microsomes of LTC4S-expressing HEK293 cells were pretreated with diflapolin, MK886 (10 µM), or vehicle for 10 min on ice with subsequent addition of LTA4-methyl ester. After 10 min at 4 °C, LTC4-methyl ester was analyzed by UPLC-MS/MS. (c) COX-1/2 activity. In cell-free assays, purified ovine COX-1 and recombinant human COX-2 were pretreated with diflapolin (10 µM), indometacin (10 µM) or vehicle (0.1% DMSO) for 5 min on ice and stimulated with AA (5 and 2 µM for COX-1 and -2, respectively for 10 min at 37 °C. 12-HHT was analyzed by HPLC. (d) Effect of diflapolin on 12- and 15-LOX. Intact neutrophils were pre- incubated by diflapolin or vehicle (0.1% DMSO) and stimulated with 2.5 µM Ca2+-ionophore plus 20 µM AA for 10 min at 37 °C. 12- and 15-HETE were determined by HPLC. (e) AA release. [3H]AA-labeled neutrophils were pretreated with diflapolin (1 µM), RSC-3388 (10 µM) or vehicle (0.1% DMSO) and stimulated by 2.5 µM Ca2+- ionophore for 15 min. Radioactivity of the supernatant was analyzed by scintillation counting. (f) Cell viability assay. Monocytes were treated with diflapolin (10 µM), staurosporin (3 µM) or vehicle (0.3% DMSO) for 24 and 48 h, respectively. Cell viability was determined by MTT assay. Data are expressed as percentage of control (100%), means + SEM, n = 3–5. *p < 0.05; **p < 0.005; ***p < 0.001 vs. vehicle control (paired t-test).

    Techniques Used: Activity Assay, Expressing, Tandem Mass Spectroscopy, Purification, Recombinant, Incubation, Labeling, Radioactivity, Viability Assay, MTT Assay, Control



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    Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and <t>LTA4-H</t> in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).
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    Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and <t>LTA4-H</t> in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).
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    Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and LTA4-H in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).

    Journal: Scientific reports

    Article Title: Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

    doi: 10.1038/s41598-017-09795-w

    Figure Lengend Snippet: Figure 2. Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and LTA4-H in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl2 for 10 min at 37 °C. LTB4 isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).

    Article Snippet: Bovine serum albumin (BSA), EDTA, glutathione, saccharose, and Tris, AppliChem (Darmstadt, Germany); L-glutamine, BioChem GmbH (Karlsruhe, Germany); β-PGE2 and tritium-labeled [5,6,8,9,11,12,14,15-3H] labeled AA, Biotrend Chemicals GmbH (Köln, Germany); AA, PGB1, 3-phenyl-cyano(6-methoxy-2-naphthalenyl) methyl ester-2-oxiraneacetic acid (PHOME), LTC4-d5-methyl ester, LTA4 methyl ester, COX isoenzymes, and MK886, Cayman Chemical (Biomol, Hamburg, Germany); acetonitrile, Dulbecco’s modified Eagle’s high glucose medium with glutamine, geneticin, penicillin/streptomycin-solution and trypsin-EDTA, GE Healthcare Life Science (Freiburg, Germany); PGH2, Larodan Fine Chemicals (Stockholm, SWE); Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 555 goat anti-mouse, hygromycin B, Lipofectamine LTX Reagent Plus, and non-immune goat serum and Sf-900TM II SFM, Invitrogen (Darmstadt, Germany).

    Techniques: Activity Assay, Recombinant, Fluorescence, Cell-Free Assay, Control, Incubation, Tandem Mass Spectroscopy

    Figure 3. Diflapolin shows target specificity within the AA cascade without cytotoxicity. (a) mPGES-1 activity. Microsomes of IL-1β-stimulated A549 cells were pretreated with diflapolin, 10 µM MK886, or vehicle for 10 min on ice and stimulated with 20 µM PGH2. After 1 min at 4 °C, PGE2 formation was analyzed by HPLC. (b) LTC4S activity. Microsomes of LTC4S-expressing HEK293 cells were pretreated with diflapolin, MK886 (10 µM), or vehicle for 10 min on ice with subsequent addition of LTA4-methyl ester. After 10 min at 4 °C, LTC4-methyl ester was analyzed by UPLC-MS/MS. (c) COX-1/2 activity. In cell-free assays, purified ovine COX-1 and recombinant human COX-2 were pretreated with diflapolin (10 µM), indometacin (10 µM) or vehicle (0.1% DMSO) for 5 min on ice and stimulated with AA (5 and 2 µM for COX-1 and -2, respectively for 10 min at 37 °C. 12-HHT was analyzed by HPLC. (d) Effect of diflapolin on 12- and 15-LOX. Intact neutrophils were pre- incubated by diflapolin or vehicle (0.1% DMSO) and stimulated with 2.5 µM Ca2+-ionophore plus 20 µM AA for 10 min at 37 °C. 12- and 15-HETE were determined by HPLC. (e) AA release. [3H]AA-labeled neutrophils were pretreated with diflapolin (1 µM), RSC-3388 (10 µM) or vehicle (0.1% DMSO) and stimulated by 2.5 µM Ca2+- ionophore for 15 min. Radioactivity of the supernatant was analyzed by scintillation counting. (f) Cell viability assay. Monocytes were treated with diflapolin (10 µM), staurosporin (3 µM) or vehicle (0.3% DMSO) for 24 and 48 h, respectively. Cell viability was determined by MTT assay. Data are expressed as percentage of control (100%), means + SEM, n = 3–5. *p < 0.05; **p < 0.005; ***p < 0.001 vs. vehicle control (paired t-test).

    Journal: Scientific reports

    Article Title: Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

    doi: 10.1038/s41598-017-09795-w

    Figure Lengend Snippet: Figure 3. Diflapolin shows target specificity within the AA cascade without cytotoxicity. (a) mPGES-1 activity. Microsomes of IL-1β-stimulated A549 cells were pretreated with diflapolin, 10 µM MK886, or vehicle for 10 min on ice and stimulated with 20 µM PGH2. After 1 min at 4 °C, PGE2 formation was analyzed by HPLC. (b) LTC4S activity. Microsomes of LTC4S-expressing HEK293 cells were pretreated with diflapolin, MK886 (10 µM), or vehicle for 10 min on ice with subsequent addition of LTA4-methyl ester. After 10 min at 4 °C, LTC4-methyl ester was analyzed by UPLC-MS/MS. (c) COX-1/2 activity. In cell-free assays, purified ovine COX-1 and recombinant human COX-2 were pretreated with diflapolin (10 µM), indometacin (10 µM) or vehicle (0.1% DMSO) for 5 min on ice and stimulated with AA (5 and 2 µM for COX-1 and -2, respectively for 10 min at 37 °C. 12-HHT was analyzed by HPLC. (d) Effect of diflapolin on 12- and 15-LOX. Intact neutrophils were pre- incubated by diflapolin or vehicle (0.1% DMSO) and stimulated with 2.5 µM Ca2+-ionophore plus 20 µM AA for 10 min at 37 °C. 12- and 15-HETE were determined by HPLC. (e) AA release. [3H]AA-labeled neutrophils were pretreated with diflapolin (1 µM), RSC-3388 (10 µM) or vehicle (0.1% DMSO) and stimulated by 2.5 µM Ca2+- ionophore for 15 min. Radioactivity of the supernatant was analyzed by scintillation counting. (f) Cell viability assay. Monocytes were treated with diflapolin (10 µM), staurosporin (3 µM) or vehicle (0.3% DMSO) for 24 and 48 h, respectively. Cell viability was determined by MTT assay. Data are expressed as percentage of control (100%), means + SEM, n = 3–5. *p < 0.05; **p < 0.005; ***p < 0.001 vs. vehicle control (paired t-test).

    Article Snippet: Bovine serum albumin (BSA), EDTA, glutathione, saccharose, and Tris, AppliChem (Darmstadt, Germany); L-glutamine, BioChem GmbH (Karlsruhe, Germany); β-PGE2 and tritium-labeled [5,6,8,9,11,12,14,15-3H] labeled AA, Biotrend Chemicals GmbH (Köln, Germany); AA, PGB1, 3-phenyl-cyano(6-methoxy-2-naphthalenyl) methyl ester-2-oxiraneacetic acid (PHOME), LTC4-d5-methyl ester, LTA4 methyl ester, COX isoenzymes, and MK886, Cayman Chemical (Biomol, Hamburg, Germany); acetonitrile, Dulbecco’s modified Eagle’s high glucose medium with glutamine, geneticin, penicillin/streptomycin-solution and trypsin-EDTA, GE Healthcare Life Science (Freiburg, Germany); PGH2, Larodan Fine Chemicals (Stockholm, SWE); Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 555 goat anti-mouse, hygromycin B, Lipofectamine LTX Reagent Plus, and non-immune goat serum and Sf-900TM II SFM, Invitrogen (Darmstadt, Germany).

    Techniques: Activity Assay, Expressing, Tandem Mass Spectroscopy, Purification, Recombinant, Incubation, Labeling, Radioactivity, Viability Assay, MTT Assay, Control

    The effect of AA-signalling agonists and antagonists on the viability of SH-SY5Y neuroblastoma cells treated with ATRA (dose-response) in combination with fixed doses of the 5-LO inhibitors celecoxib or MK886.

    Journal: PLoS ONE

    Article Title: Cell Survival Signalling through PPARδ and Arachidonic Acid Metabolites in Neuroblastoma

    doi: 10.1371/journal.pone.0068859

    Figure Lengend Snippet: The effect of AA-signalling agonists and antagonists on the viability of SH-SY5Y neuroblastoma cells treated with ATRA (dose-response) in combination with fixed doses of the 5-LO inhibitors celecoxib or MK886.

    Article Snippet: All- trans RA (ATRA), AACOCF3, GSK0660, MK886 and Prostaglandin E2 (PGE2) were from Sigma-Aldrich, PD-146176 from Enzo Life Sciences (Farmindale, NY), celecoxib from Pfizer (NY), baicalein, 5-oxo-ETE and leukotriene A4 (LTA4) methyl ester were from Cayman Chemicals (Ann Arbor, MI).

    Techniques: