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: 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 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:

Article Title: Comparative Analysis of Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Activity in Serum and Lethality in Mice and Rabbits Pretreated with the Staphylococcal Superantigen Toxic Shock Syndrome Toxin 1

doi: 10.1128/IAI.69.11.7169-7172.2001

Figure Lengend Snippet: Dose response of LPS-induced lethality and TNF-α activity in serum in rabbits primed with TSST-1

Article Snippet: When measured in the bioassay for TNF-α, the specific activity of murine recombinant TNF-α (R & D systems) was 2.56 × 10 8 U/mg, while that of rabbit TNF-α in conditioned medium (Pharmingen) was 3.72 × 10 8 U/mg.

Techniques: Activity Assay

Journal:

Article Title: Comparative Analysis of Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Activity in Serum and Lethality in Mice and Rabbits Pretreated with the Staphylococcal Superantigen Toxic Shock Syndrome Toxin 1

doi: 10.1128/IAI.69.11.7169-7172.2001

Figure Lengend Snippet: Time course of LPS-induced TNF-α in serum in Dutch belted rabbits primed with TSST-1. Groups of three rabbits were injected with 10.0 ng (i.v.) of TSST-1 (primed)/kg or an equivalent volume of PBS (unprimed). All rabbits were injected with LPS (10 μg/kg [i.v.]) 12 h later. Control rabbits injected with 5 μg of TSST-1 and PBS/kg 12 h later did not develop detectable levels of TNF-α. (∗, P ≤ 0.05).

Article Snippet: When measured in the bioassay for TNF-α, the specific activity of murine recombinant TNF-α (R & D systems) was 2.56 × 10 8 U/mg, while that of rabbit TNF-α in conditioned medium (Pharmingen) was 3.72 × 10 8 U/mg.

Techniques: Injection, Control

Journal:

Article Title: Comparative Analysis of Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Activity in Serum and Lethality in Mice and Rabbits Pretreated with the Staphylococcal Superantigen Toxic Shock Syndrome Toxin 1

doi: 10.1128/IAI.69.11.7169-7172.2001

Figure Lengend Snippet: Direct comparison of LPS-induced TNF-α in serum in mice and rabbits primed with TSST-1. Groups of three BALB/c-AnNCr mice and three Dutch belted rabbits were injected with TSST-1 at doses of 200 μg/kg (i.p.) or 10.0 ng/kg (i.v.), respectively. After 12 h, mice and rabbits were injected with LPS at doses of 400 μg/kg (i.p.) or 10 μg/kg (i.v.), respectively. TNF-α activity in serum was measured at 2 h postinjection of LPS in both species. The levels of TNF-α due to LPS alone were calculated from previous time course studies with the same mice and rabbits. Injection of TSST-1 alone did not induce detectable levels of TNF-α in serum in control animals. The mean peak TNF-α level induced by TSST-1 plus LPS in mice did not significantly differ from that measured in rabbits.

Article Snippet: When measured in the bioassay for TNF-α, the specific activity of murine recombinant TNF-α (R & D systems) was 2.56 × 10 8 U/mg, while that of rabbit TNF-α in conditioned medium (Pharmingen) was 3.72 × 10 8 U/mg.

Techniques: Comparison, Injection, Activity Assay, 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: 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