Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Overexpression of TRIM38 inhibits TNFα- and IL-1β–triggered signaling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) or left untreated for 10 h before luciferase assays were performed. Expression of transfected TRIM38 in each unstimulated sample was examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HCT116 and HeLa cells. The experiments were performed as in A. (C) Effects of TRIM38 on IFNγ-induced activation of the IRF1 promoter. The experiments were performed as in A except that the IRF1 promoter reporter plasmid was used and transfected cells were treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of TRIM38 in the stable cell lines was examined by immunoblot analysis (Right). (E) Effects of TRIM38 on TNFα- and IL-1β–induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then the medium was collected for ELISA analysis. (F) Effects of TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Transduction, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β–triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then total RNA was extracted for qPCR analysis. (E) Effects of TRIM38 deficiency on TNFα and IL-1β–induced cytokine production. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then the medium was collected for ELISA analysis. (F) TRIM38 deficiency potentiates TNFα-triggered MAPK activation. The indicated cells (1 × 107) were left untreated or treated with TNFα (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis was performed with the indicated antibodies. (G) TRIM38 deficiency potentiates IL-1β–triggered MAPK activation. The experiments were performed as in E, except that cells were treated with IL-1β (10 ng/mL). Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Knockdown, Knock-Out, Transfection, Expressing, Western Blot, Control, Activation Assay, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY-SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian overexpression system. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (B) Endogenous TRIM38 interacts with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h, and then immunoblots were performed with the indicated antibodies. (D) Effects of TRIM38 truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression in TRIM38−/− cells stably transduced with an empty vector (II), TRIM38-Flag (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and in TRIM38+/+ cells stably transduced with an empty vector (I). Cells (1 × 107) (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 mutant (63–465) into TRIM38-deficient cells leads to down-regulation of TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or IL-1β for the indicated times. Immunoblot analysis was performed with the indicated antibodies.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 × 105) were transfected with the indicated plasmids. Fourteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (B) Effects of NH4Cl and MG132 on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) cells were treated with NH4Cl or MG132 for 4 h and then further treated with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) were transfected with Cherry-TAB2 and GFP-LAMP1 (Left) or CFP-TRIM38 (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) paraformaldehyde and subjected for confocal microscopy. (D) Effect of TRIM38 deficiency on TNFα- or IL-1β–induced colocalization of TAB2 with the lysosomes. TRIM38+/+, TRIM38−/−, or TRIM38−/− cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) paraformaldehyde and subjected to confocal microscopy. A random 10 cells in each sample were used for calculating the colocalization dots that were normalized to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Translocation Assay, Confocal Microscopy, Staining

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 1. Ad vectors containing TNFa expression cassettes. Both Ad-75 (Ad- mTNF-75) and Ad-hTNF (Ad-MCMV- TNF)25 vectors express the mature mTNFa and hTNFa cDNAs, respec- tively, fused with the IFN-g signal pep- tide. Ad-mTNF (Ad-mTNF-wt)24 con- tains the entire mTNFa cDNA. All TNFa-expressing vectors use the MCMV promoter and an SV40 polyad- enylation signal. All vectors are E1/E3- deleted Ad-5-based vectors.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: Expressing

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 2. TNF expression in Ad-vector-transduced cell supernatants: detection of mutant (D142N-A144R) mTNFa expression by Western blot with a polyclonal goat anti-mouse TNFa Ab. Expression was detected from the supernatants of the Ad-mTNF- or Ad-75-transduced MRC5, NDL, and MT1A2 (1A2) cell lines as described in Materials and Methods. Samples (12.5 mL of supernatant per well) were mixed 50:50 with loading buffer. Ad-BHGDE1,3-transduced MT1A2 cells were used as a negative control.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Negative Control

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 3. In vitro bioactivity of mutant mTNFa. A: Cytotoxicity (MTT) assay on TNFa-sensitive L929 cells with 10 mL of supernatants from Ad-75- (75), Ad-mTNF- (mTNF), Ad-hTNF- (hTNF), and Ad-BHGDE1,3- (E1,3) transduced MRC5, NDL, and MT1A2 cells (MOI 5 50 PFU/cell). The cytotoxicity of standard dilutions of recombinant mTNFa is indicated by the line graph on the right. B: Proliferation assay on the TNFa-responsive CT6 cell line (detected by MTT), treated with 10 mL of supernatants from Ad vector-transduced MT1A2 cells (MOI 5 50 PFU/cell). Solid bars represent wells treated with soluble murine p55 TNFR (33 mg/mL). The proliferation of CT6 cells in response to recombinant mTNFa is indicated by the cross-hatched bars on the right.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: In Vitro, Mutagenesis, MTT Assay, Recombinant, Proliferation Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

doi: 10.1038/sj.cgt.7700068

Figure Lengend Snippet: Figure 4. In vitro binding of mutant and wt TNFa to TNFR: coimmunoprecipitation/Western blot of wt hTNFa and mTNFa and mutant (D142N-A144R) mTNFa in 500 mL of culture supernatant from NDL cells transduced with the respective Ad vectors (MOI 5 50 PFU/cell) using soluble murine p55 and p75 TNFRs (2 mg per precipitation), as described in Materials and Methods. Receptors were immunoprecipitated by specific Abs that did not interfere with ligand binding (2 mg per precipitation). Coimmunoprecipitates were analyzed by Western blot, first for mTNFa, and second for hTNFa (see Materials and Methods). sTNFR refers to soluble TNFR.

Article Snippet: Western blotting was done by blocking the membrane with 5% powdered milk in PBS and subsequently incubating with primary polyclonal goat anti-mouse TNFa Ab (R&D Systems) for 4 hours at room temperature or overnight at 4°C.

Techniques: In Vitro, Binding Assay, Mutagenesis, Western Blot, Transduction, Immunoprecipitation, Ligand Binding Assay

Journal: Cancer Research

Article Title: Adaptive Upregulation of EGFR Limits Attenuation of Tumor Growth by Neutralizing IL6 Antibodies, with Implications for Combined Therapy in Ovarian Cancer

doi: 10.1158/0008-5472.can-14-1801

Figure Lengend Snippet: Figure 5. Effects of inhibition of IL6 and/or ErbB-1 in mouse ovarian cancer xenografts. A, weight of dissected peritoneal tumors collected from mice injected intraperitoneally with IGROV-1 and treated with either an IgG antibody, an anti-IL6 antibody, gefitinib, or their combination. Data in A are shown as mean SEM of two independent experiments, with a total of 8 mice per experimental group in each experiment. Statistical analysis was performed and is shown as , P 0.05; , P 0.01; , P 0.001. B, pSTAT3, pERK, and pErbB-1 staining on mouse tumor sections from the above experiment. C, ErbB-1 staining on mouse tumor sections. D, Western blot analysis and quantification of pSTAT3, pErbB-1, and pERK in protein extracts from mouse xenografts treated with an anti-IL6 antibody. E, Western blot analysis and quantification of TNFa and Jagged1 in protein extracts from mouse xenografts treated with an anti-IL6 antibody. Quantification was done using ImageJ software and is always relative to the total protein. Results represented on graphs correspond to fold increase.

Article Snippet: The membrane was blocked overnight (4 C in PBS with 0.1% Tween and 5% milk powder) and probed using the following antibodies: PhosphoStat3 (Tyr705; 1:1,000 #9145, Cell Signaling Technology), Stat3 (1:1,000 #4904, Cell Signaling Technology), p-ERK (1:1,000 sc-7383, Santa Cruz Biotechnology), ERK (1:1,000 #9102, Cell Signaling Technology), phospho-ErbB-1 (1:1,000 #2220, Cell Signaling Technology), ErbB-1 (1:1,000 2232, Cell Signaling Technology), phospho-ErbB-4 (1:500 #3790, Cell Signaling Technology), ERbB-4 (1:1,000 #4795, Cell Signaling Technology), TNFa (1:1,000 ab6671, Abcam), Jagged1 (1:1,000 SC-8303, Santa Cruz Biotechnology), a-tubulin (1:2,000 sc-8035, Santa Cruz Biotechnology), lamin A/C (1:2,000 #2032, Cell Signaling Technology), b-actin (1:5,000 A5316, Sigma).

Techniques: Inhibition, Injection, Staining, Western Blot, Software