Journal: Hepatology Communications

Article Title: Proteomics‐based identification of the role of osteosarcoma amplified‐9 in hepatocellular carcinoma recurrence

doi: 10.1002/hep4.1952

Figure Lengend Snippet: Differential proteins expression in OS‐9 overexpression group (n = 3) and vector control (n = 3) of SMMC‐7721 cells. (A) Volcano plot of the differentially expressed proteins. Gray dots represent genes that are not differentially expressed in the early recurrence group and nonrecurrence group; red dots and blue dots represent genes that are up‐regulated and down‐regulated significantly in the early recurrence group. (B) Heat map of the differentially expressed proteins. Red rectangles mean that genes are up‐regulated in these samples, and blue ones mean down‐regulated. Two hundred and sixty‐eight protein expressions were up‐regulated and 140 protein expressions were down‐regulated in the overexpression group compared with the vector control group (fold change ≥ 1.5; p < 0.05). (C) Heat map of the differentially expressed proteins classified to the hypoxia‐inducible factor 1 (HIF‐1) and tumor necrosis factor (TNF) signaling pathway. (D) Ridgeline plot of Kyoto Encyclopedia of Genes and Genomes pathway enrichment for differentially expressed proteins. (E) Gene Ontology functions for differentially expressed proteins. The left side of the circle includes all related genes, and the right side displays the Gene Ontology terms. Red and blue rectangles mean that genes are up‐regulated and down‐regulated in the early recurrence group. Abbreviations: ALDOA, aldolase, fructose‐bisphosphate A; ALDOC, aldolase, fructose‐bisphosphate C; BCL10, BCL10 immune signaling adaptor; CASP7, caspase 7; CCNB1, cyclin B1; CDK6, cyclin dependent kinase 6; CEBPB, CCAAT enhancer binding protein beta; CHEK2, checkpoint kinase 2; CREBBP, CREB binding protein; DDX58, DExD/H‐box helicase 58; ENO1, enolase 1; ENO2, enolase 2; ENO3, enolase 3; HK2, hexokinase 2; HMOX1, heme oxygenase 1; IGFBP3, insulin like growth factor binding protein 3; JUNB, JunB proto‐oncogene; LDHA, lactate dehydrogenase A; MALT1, MALT1 paracaspase; MLKL, mixed lineage kinase domain like pseudokinase; OE, overexpressed; PGK1, phosphoglycerate kinase 1; PLCG2, phospholipase C gamma 2; SERPINE1, serpin family E member 1; SFN, stratifin. NC, control; STEAP3, STEAP3 metalloreductase; TNFAIP3, TNF alpha induced protein 3; TRAF5, TNF receptor associated factor 5

Article Snippet: Inhibitors including HIF‐1α and tumor necrosis factor alpha (TNF‐α) were obtained (VH‐298, MedChemExpress; HY‐100947, Methylthiouracil; HY‐B0513, MedChemExpress).

Techniques: Expressing, Over Expression, Plasmid Preparation, Control, Binding Assay

Journal: Hepatology Communications

Article Title: Proteomics‐based identification of the role of osteosarcoma amplified‐9 in hepatocellular carcinoma recurrence

doi: 10.1002/hep4.1952

Figure Lengend Snippet: (A–K) The relative messenger RNA (mRNA) expression level of lineage kinase domain‐like MLKL (A), tumor necrosis factor alpha–induced protein 3 (TNFAIP3) (B), JunB proto‐oncogene (JUNB) (C), and tumor necrosis factor receptor–associated factor 5 (TRAF5) (D), TNF‐α (E) and caspase‐7 (CASP7) (F) could be observed to increase more than 2‐fold ( p < 0.01). The relative expression of enolase1 (ENO1) (G), enolase2 (ENO2) (H), enolase3 (ENO3) (I), aldolase, fructose‐bisphosphate A (ALDOA) (J), and lactate dehydrogenase A (LDHA) (K) were also elevated more than 2‐fold ( p < 0.01)

Article Snippet: Inhibitors including HIF‐1α and tumor necrosis factor alpha (TNF‐α) were obtained (VH‐298, MedChemExpress; HY‐100947, Methylthiouracil; HY‐B0513, MedChemExpress).

Techniques: Expressing

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

Journal: Oncotarget

Article Title: PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

doi: 10.18632/oncotarget.6955

Figure Lengend Snippet: ( A ) Scatter plot illustrating TNF-α gene expression in prostate cancer patient samples classified by PTEN mRNA levels. ( B ) Bar graph showing TNF-α secretion from PTEN-depleted Sh11.02 cells following treatment with a single 3 Gy dose of IR. ( C ) Immunoblot demonstrating the impact of 3 Gy IR on TNFR-1 expression in Sh11.02 cells. ( D ) Bar graph showing TNF-α secretion levels from THP-1 cells following treatment with 3 Gy IR. ( E ) Bar graph showing TNF-α secretion levels from THP-1 cells following treatment with 3 nM CXCL8. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t -test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Recombinant human TNF-α was purchased from Alomone Labs (Jerusalem, Israel) and cells were treated at a final concentration of 10 ng/ml.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Oncotarget

Article Title: PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

doi: 10.18632/oncotarget.6955

Figure Lengend Snippet: ( A ) Bar graph illustrating luciferase reporter assay analysis of NFκB activity in Sh11.02 cells. Different experimental conditions included THP-1 co-culture, exposure to 3 Gy IR and treatment with a TNF-α neutralizing antibody (10 ng/ml). ( B ) Immunoblot showing basal expression of NFκB-regulated anti-apoptotic targets in PTEN-modulated DU145 populations. ( C ) Immunoblot showing the effect of 10 ng/ml recombinant TNF-α treatment on expression of NFκB-regulated anti-apoptotic targets in Sh11.02 cells. ( D ) Immunoblot illustrating expression of NFκB targets in Sh11.02 cells 4 h following 3 Gy IR in the presence or absence of THP-1 co-culture and TNF-α neutralizing antibody. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences in luciferase assay results were determined by performing a two-tailed Students t -test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Recombinant human TNF-α was purchased from Alomone Labs (Jerusalem, Israel) and cells were treated at a final concentration of 10 ng/ml.

Techniques: Luciferase, Reporter Assay, Activity Assay, Co-Culture Assay, Western Blot, Expressing, Recombinant, Two Tailed Test

Journal: Oncotarget

Article Title: PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

doi: 10.18632/oncotarget.6955

Figure Lengend Snippet: ( A ) Immunoblot showing cIAP-1 expression in Sh11.02 cells following THP-1 co-culture, 3 Gy IR and treatment with 0.1 μM AT-IAP. Equal protein loading was confirmed by re-probing for GAPDH. ( B ) Bar graph showing caspase 3/7 activity in Sh11.02 cells following THP-1 co-culture, 3 Gy IR and treatment with 0.1 μM AT-IAP for 6 h. ( C ) Clonogenic survival curve showing the radiosensitizing potential of AT-IAP on Sh11.02 cells with THP-1 co-culture. Data showing addition of a TNF-α neutralizing antibody in this system is also presented. Data shown is the mean plus or minus standard error of the mean value, calculated from a minimum of three independent experiments. Statistically significant differences were determined by performing a two-tailed Students t -test or two-way ANOVA for clonogenic assays (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Recombinant human TNF-α was purchased from Alomone Labs (Jerusalem, Israel) and cells were treated at a final concentration of 10 ng/ml.

Techniques: Western Blot, Expressing, Co-Culture Assay, Activity Assay, Two Tailed Test