Journal: The FASEB Journal

Article Title: Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium

doi: 10.1096/fj.201802608R

Figure Lengend Snippet: The Crth2 antagonist CAY10595 up-regulates inflammatory gene expression during LPS stimulation of BMDMs. A–C) Representative immunoblot (A) and quantitation of Cox2 (B) and iNOS (C) levels in BMDMs treated with DMSO or CAY10595 (5 µM) after 4–24 h LPS stimulation (100 ng/ml) or no stimulation (0 h). β-actin is used as loading control; n = 3 independent experiments. D) Mean fold change of mRNA levels of Cox2 (4 h), iNOS (4 h), mPges1 (8 h), Tnf-α (4 h), and Il-1β (4 h) in BMDMs treated with CAY10595 relative to BMDMs treated with 0.1% DMSO after LPS stimulation (100 ng/ml); n = 4–5 independent experiments. E) PGE2 production 8 h after LPS stimulation; n = 3 independent experiments. F) Nitrite production 4–24 h after LPS stimulation; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with DMSO (Student’s t test).

Article Snippet: Treatments Various compounds such as antagonists, agonists, and inhibitors were used at the following concentrations: 100 ng/ml LPS ( Escherichia coli serotype 0128:B12; MilliporeSigma); 10 mg/kg i.p. zymosan A (MilliporeSigma); 10 ng/ml Tnf-α (mouse Tnf-α; GoldBio, St. Louis, MO, USA); 5 μM CAY10595, 25 µM HQL79, 30 mM H89, and 20 nM 13,14-dihydro-15-keto-PGD 2 (DKPGD 2 ) (all from Cayman Chemicals, Ann Arbor, MI, USA); and 1 µg/ml pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, USA).

Techniques: Expressing, Western Blot, Quantitation Assay

Journal: The FASEB Journal

Article Title: Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium

doi: 10.1096/fj.201802608R

Figure Lengend Snippet: Deletion of Crth2 in murine macrophages increases inflammatory gene expression. A, B) Representative immunoblot (A) and quantitation (B) of iNOS and Cox2 in Crth2−/− and NTC RAW264.7 cells after 8 h of LPS stimulation (100 ng/ml). β-Actin is used as loading control. P value is in comparison with NTC RAW264.7 cells; n = 3 independent experiments. C) Mean fold change of mRNA levels of Cox2, iNOS, and mPges1 in Crth2−/− RAW264.7 cells relative to NTC RAW264.7 cells after LPS stimulation for 8 h. P value is in comparison with NTC RAW264.7 cells; n = 3–5 independent experiments. D) Nitrite production after 8 h after LPS stimulation. n = 5 independent experiments. E) PGE2 production after 8 h of LPS stimulation; n = 3 independent experiments. F) Densitometry of expression of Cox2 in NTC and Crth2−/− RAW264.7 cells after 8 h of Tnf-α stimulation (10 ng/ml). Representative immunoblot is in the inset. β-actin was used as loading control. P value is in comparison with NTC RAW264.7 cells; n = 3 independent experiments. G) PGE2 production after 8 h of Tnf-α stimulation; n = 3 independent experiments. WT and Crth2−/− mice were treated with zymosan A (10 mg/kg body weight) for 24 h. Peritoneal lavage fluid was obtained from mice after zymosan treatment, and cells were isolated by centrifugation. Pelleted cells were used for mRNA as described in Materials and Methods. H) Mean fold change in mRNA levels of Cox2 and iNOS in peritoneal lavage cells isolated from WT and Crth2−/− mice. P value is in comparison with the expression recorded in WT lavage cells; n = 5 independent experiments. I–K) Immunoblot (I) and quantitation of iNOS (J) and Cox2 (K) in Crth2+/− and WT BMDMs after 8 h of LPS stimulation. β-actin is used as loading control. P value is in comparison with WT BMDMs; n = 3–5 independent experiments. L) Mean fold change of mRNA levels of iNOS and Cox2 in Crth2+/− BMDMs after 8 and 12 h of LPS stimulation relative to WT. P value is in comparison with WT BMDMs. M) Mean fold change of mRNA levels of iNOS and Cox2 in Crth2+/− BMDMs treated with CAY10595 after 12 h of LPS stimulation relative to Crth2+/− BMDMs treated with 0.1% DMSO. P value is in comparison with DMSO-treated Crth2+/− BMDMs. Technical triplicate of RNA pooled from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test).

Article Snippet: Treatments Various compounds such as antagonists, agonists, and inhibitors were used at the following concentrations: 100 ng/ml LPS ( Escherichia coli serotype 0128:B12; MilliporeSigma); 10 mg/kg i.p. zymosan A (MilliporeSigma); 10 ng/ml Tnf-α (mouse Tnf-α; GoldBio, St. Louis, MO, USA); 5 μM CAY10595, 25 µM HQL79, 30 mM H89, and 20 nM 13,14-dihydro-15-keto-PGD 2 (DKPGD 2 ) (all from Cayman Chemicals, Ann Arbor, MI, USA); and 1 µg/ml pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, USA).

Techniques: Expressing, Western Blot, Quantitation Assay, Isolation, Centrifugation

Journal: The FASEB Journal

Article Title: Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium

doi: 10.1096/fj.201802608R

Figure Lengend Snippet: TNF-α is up-regulated by the miR-155–Carhsp1 axis in Crth2−/− RAW264.7 cells. A) Mean fold change of mRNA levels of miR-155 in NTC and Crth2−/− RAW264.7 cells after 8 h of LPS stimulation (100 ng/ml); n = 6 independent experiments. B, C) Representative immunoblot (B) and densitometric quantitation (C) of Carhsp1 levels in NTC and Crth2−/− RAW264.7 cells after 8 h LPS stimulation (100 ng/ml). β-Actin is used as loading control; n = 4 independent experiments. D) Tnf-α production 1 h after LPS stimulation. n = 3 independent experiments; n = 3 independent experiments. E) Mean fold change of mRNA levels of Carhsp1 8 h after LPS stimulation relative to NTC RAW264.7 cells; n = 3 independent experiments. *P < 0.05, ****P < 0.0001 compared with NTC (Student’s t test).

Article Snippet: Treatments Various compounds such as antagonists, agonists, and inhibitors were used at the following concentrations: 100 ng/ml LPS ( Escherichia coli serotype 0128:B12; MilliporeSigma); 10 mg/kg i.p. zymosan A (MilliporeSigma); 10 ng/ml Tnf-α (mouse Tnf-α; GoldBio, St. Louis, MO, USA); 5 μM CAY10595, 25 µM HQL79, 30 mM H89, and 20 nM 13,14-dihydro-15-keto-PGD 2 (DKPGD 2 ) (all from Cayman Chemicals, Ann Arbor, MI, USA); and 1 µg/ml pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, USA).

Techniques: Western Blot, Quantitation Assay

Journal: The FASEB Journal

Article Title: Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages via changes in intracellular calcium

doi: 10.1096/fj.201802608R

Figure Lengend Snippet: Schematic representation of the Crth2-dependent control of NF-κB gene expression in macrophages. Cyclopentenone metabolites of PGD2, Δ12-PGJ2 and 15d-PGJ2, are produced during LPS-induced inflammation in murine macrophages. Crth2 activation by CyPGs or synthetic ligands such as DKPGD2 increases Ca2+ influx through SOCE. The Gαi signals down-regulate Carhsp1, which stabilizes Tnf-α mRNA via increased miR-155 expression to decrease inflammatory responses triggered through the TNF-α–NF-κB axis. Additionally, Crth2 signals inhibit adenylate cyclase activity to suppress PKA-dependent activation of NF-κB to down-regulate LPS-induced inflammatory response. hPgds, hematopoietic prostaglandin D synthase.

Article Snippet: Treatments Various compounds such as antagonists, agonists, and inhibitors were used at the following concentrations: 100 ng/ml LPS ( Escherichia coli serotype 0128:B12; MilliporeSigma); 10 mg/kg i.p. zymosan A (MilliporeSigma); 10 ng/ml Tnf-α (mouse Tnf-α; GoldBio, St. Louis, MO, USA); 5 μM CAY10595, 25 µM HQL79, 30 mM H89, and 20 nM 13,14-dihydro-15-keto-PGD 2 (DKPGD 2 ) (all from Cayman Chemicals, Ann Arbor, MI, USA); and 1 µg/ml pertussis toxin (PTX; List Biological Laboratories, Campbell, CA, USA).

Techniques: Expressing, Produced, Activation Assay, Activity Assay