Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: The Journal of Pathology

Article Title: Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

doi: 10.1002/path.4439

Figure Lengend Snippet: Double minute chromosome (DM)-containing tumour cells often show constitutive activation of the ERK1/2–MAPK pathway. (A, F) The number of DMs/cell in various human malignant tumour cells (bars represent mean ± SD). (B–D, G) Phosphorylation status of ERK1/2, p38 and JNK1/2/3 in DM-containing tumour cells; phosphorylation of ERK1/2, p38 and JNK1/2/3 in MEKK3- or p38-over-expressing or TNF α -treated HEK293T cells (10 ng/ml for 30 min) are shown as positive controls. (E) Microarray analysis of UACC-1598DM compared to UACC-1598HSR cells

Article Snippet: Recombinant human TNF α was purchased from BioVision (Mountain View, CA, USA).

Techniques: Activation Assay, Expressing, Microarray

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Lentiviral vector mediated delivery and expression of sTNFR-Fc . (A) Schematic maps of the lentiviral transfer plasmids, pHR-hTNFR-Fc-eGFP and pHR-Fc-eGFP. LTR: long terminal repeat; ψ: packaging signal; SA, SD: splice donor, splice acceptor; CMV: cytomegalovirus promoter; hTNFR-Fc: codon-optimized gene encoding the extracellular domain of the human TNF receptor type 2, fused to the hinge domain from IgG1 and the Fc domain from human IgG3; Fc: hinge domain from IgG1 and the Fc domain from human IgG3; IRES: internal ribosome entry site; GFP: green fluorescent protein.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Stable and high level secretion of sTNFR-Fc in transduced cells . (A) Immunoblot detection of sTNFR-Fc protein in transfected cells. Mock = supernatant from mock transfected 293T cells; Intracellular = transfected cell lysate; and Secreted = supernatant from the transfected 293T cells. (B) Immunoblot detection of sTNFR-Fc protein in transduced cells intracellularly (cell lysate) and extracellularly (supernatant). HTB-T = transduced HTB-11 cells; CHME-T2 = CHME-5 cells transduced twice with the vector; CHME-T1 = CHME-5 cells transduced once with the vector. (C) Stable expression of sTNFR-Fc protein in conditioned medium of transduced cells. Expression of sTNFR-Fc in supernatants from transduced cells was measured by ELISA; results shown at different cell passages represent mean values from three independent experiments and error bars denote the standard deviation. (D) Stable expression of GFP in lentivirus vector-transduced cells. Transduced cells were passed in vitro and the percentage of GFP positive cells was determined every 5 passages by calculating the percentage of GFP+ cells within the culture using an inverted fluorescent microscope (Nikon Eclipse TE2000-U) with a digital camera attachment. Data presented here represent mean values from at least three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Microscopy

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Specific binding of expressed sTNFR-Fc to TNF-α by dot immunoblot assay . One microgram of standard recombinant human TNF-α protein was spotted onto nitrocellular membrane (NCM) and then blocked and incubated with supernatants from normal and transduced cells. After three washes with TBST, a goat-anti-human IgG Fc-HRP conjugate was applied and incubated at RT for 1 h. Specific binding was visualized by color deposition on the NCM following incubation with diaminobenzidine (DAB) substrate. Lanes: HTB-NT = supernatant from non-transduced HTB-11 cells and CHME-NT = supernatant from non-transduced CHME-5 cells used as negative controls; HTB-T = supernatant collected from transduced HTB-11 cells and CHME-T2 = supernatant from transduced CHME-5 cells; and C + = purified recombinant sTNFR-Fc protein as a positive control.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Binding Assay, Western Blot, Recombinant, Membrane, Incubation, Purification, Positive Control

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Functional antagonization of sTNFR-Fc against TNF-α . As described in materials and methods, TNF-α sensitive L929 cells were treated with TNF-α alone (80 ng/mL) or with TNF-α plus culture supernatants from vector-transduced cells; purified recombinant sTNFR-Fc protein (160 ng/mL) was used as a positive control. After incubation for 24 h, cell viability was then evaluated by MTT assay. Viability was significantly higher for the cells treated with conditioned medium from transduced cells expressing hTNFR-Fc (** p < 0.01 for HTB-T; *p < 0.05 for CHME-T) when compared to cultures that received TNF-α alone, or TNF-α plus culture supernatants from parental cells (CHME-N and HTB-N). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Functional Assay, Plasmid Preparation, Purification, Recombinant, Positive Control, Incubation, MTT Assay, Expressing, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: sTNFR-Fc mediated protection of neuronal cells from TNF-α, HIV-Tat and gp120 . (A) Non-transduced human neuronal cells, HTB-11, were treated with TNF-α alone (80 ng/mL), or with TNF-α plus culture supernatants (1:10 dilution) from vector-transduced cells (as indicated). Cells were incubated at 37°C for 3 days, and viability of the cells was determined by the trypan blue exclusion assay. sTNFR-Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the hTNFR-Fc encoding vector; Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the vector encoding human Fc only; rTNFR = purified commercial recombinant TNFR-Fc protein (160 ng/mL), used as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (B) Vector transduced human HTB-11 cells were exposed to TNF-α as described in 7A. HTB-11 cells transduced either with the sTNFR-Fc or Fc encoding vector were used in this experiment, along with non-transduced cells as controls. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (C) sTNFR-Fc protects primary rat neurons against HIV-1 Tat-mediated toxicity. Medium from vector-transduced or parental HTB-11 cells was collected at day 10, diluted and mixed with 500 nM HIV-1 Tat protein, prior to addition to primary rat neurons. Following 24 hours incubation at 37°C, these test cultures along with the control were analyzed by TUNEL assay. Cell death was significantly reduced in neuronal cultures that were treated with Tat plus conditioned medium from HTB-11 cells transduced with the sTNRF-Fc encoding lentiviral vector (HTB-T 10d) versus cells treated with Tat plus conditioned medium from parental HTB-11 cells (HTB 10d) (*p < 0.01). NT, normal neuronal cells received no Tat, and -, neuronal cells exposed to Tat as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (D) sTNFR-Fc mediated neuronal protection of human HTB-11 cells against HIV-1 gp120 toxicity. Conditioned media from hTNFR-Fc or Fc vector transduced HTB-11 cells were collected, diluted and mixed with 100 ng/mL (gp120A) or 250 ng/mL (gp120B) HIV-1 gp120 protein, with or without HIV-1 Tat, prior to addition to HTB-11 cells. Following 3 days incubation at 37°C, cell viability of these cultures, together with control cultures, was determined by trypan blue exclusion assay (***p < 0.001). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Incubation, Trypan Blue Exclusion Assay, Transduction, Purification, Recombinant, Positive Control, Standard Deviation, TUNEL Assay

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Example of Nyquist plots for the Randles equivalent circuit model obtained by analyzing cortisol standard solutions in PBS (2, 10, 15, and 50 ng mL −1 ). EIS frequency ranged from 5 Hz to 100 kHz, and a sinus amplitude of 25 mV with polarization potential of −1.5 V; ( B ) sensitivity curves obtained by analyzing standard solution containing cortisol or other HF biomarkers (e.g., TNF-α and NT-proBNP) in the concentration range 2–50 ng mL −1 using the HfO 2 substrate functionalized with anti-cortisol antibody.

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques: Concentration Assay

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Capacitance–voltage plots for cortisol detection using the capacitance biosensor; ( B ) the calibration curves of the cortisol detection (black curve) and the two interferences: TNF-α (red curve) and NT-proBNP (blue curve).

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques:

Journal: Micromachines

Article Title: A Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis

doi: 10.3390/mi13122235

Figure Lengend Snippet: ( A ) Capacitance–voltage plots for TNF-α detection; ( B ) for NT-proBNP detection using the capacitive immunosensor.

Article Snippet: Recombinant human TNF-α (Cat. No. 210-TA) was supplied by BioTechne (R&DSystems, noyal chatillon sur seiche, France).

Techniques:

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