Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Univariate analysis of the correlation between clinicopathological factors and survival time of patients with gastric cancer (n=193).

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Cell Differentiation

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Immunohistochemical analysis showing the expression of ADAM8, ADAM9, ADAM10, ADAM12 and ADAM17 in adjacent non-cancerous gastric tissues, gastric cancer tissues, positive metastatic lymph node tissues, and negative metastatic lymph node tissues. Scale bars, 50 µ M. ADAM, disintegrin and metalloproteinase domain-containing protein.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Pathological scores of ADAM8, ADAM9, ADAM10, ADAM12 and ADAM17 expression according to the staining intensity and the percentage of cell staining. (A) Pathological scores of ADAMs in adjacent non-cancerous gastric tissues (n=15) and gastric cancer tissues (n=15). (B) Pathological scores of ADAMs in positive metastatic lymph node and negative metastatic lymph node tissues (n=15). * P<0.05, ** P<0.01 vs. the corresponding control group. ADAM, disintegrin and metalloproteinase domain-containing protein.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Expressing, Staining, Control

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Relative expression of ADAM9 and ADAM17 in different tissues was examined by western blotting. (A) ADAM9 and ADAM17 expression in adjacent non-cancerous gastric tissues (n=5) and gastric cancer tissues (n=5). (B) ADAM9 and ADAM17 in positive (n=5) and negative metastatic lymph node tissues (n=5). * P<0.05, *** P<0.001 vs. the corresponding control group. ADAM, disintegrin and metalloproteinase domain-containing protein.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Multivariate survival analysis of gastric cancer (Cox proportional hazards model).

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Independent predictors of lymph node metastasis based on the multivariate logistic regression analysis.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Correlation of mortality risk scores in 193 patients. (A) The number of patients distributed across different risk score ranges. (B) Mortality incidence was correlated with the risk score. The results showed that the higher the risk scores, the greater is the risk of mortality. (C) Receiver operating characteristic curves of different predictors for mortality risk. Four predictors, including the risk score model, T staging, N staging and ADAM17 expression, were included in the analysis. ADAM, disintegrin and metalloproteinase domain-containing protein.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Analysis of AUC of different predictors for mortality risk.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Expression of ADAM17 in gastric cancer cells. (A) western blotting showed the expression of ADAM17 in BGC-823, AGS, KATO III and SGC-7901 cells. (B) ADAM17 expression in mock-transfected cells, pcDNA-3-transfected, or pcDNA-3-ADAM17-transfected BGC-823 cells. (C) ADAM17 expression in mock-transfected, siRNA-NC-transfected or siRNA-ADAM17-transfected SGC-7901 cells. All experiments were repeated three times and data are presented as the mean ± standard deviation. * P<0.05 vs. corresponding control group. ADAM, disintegrin and metalloproteinase domain-containing protein; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Expressing, Western Blot, Transfection, Standard Deviation, Control, Small Interfering RNA, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Effects of ADAM17 overexpression and suppression on cell viability and migration. (A and B) Cell Counting kit-8 assays showed the cell viability of transfected (A) BGC-823 and (B) SGC-7901 cells. (C and D) Scratch wound healing assay showed cell migration of transfected (C) BGC-823 and (D) SGC-7901 cells. All experiments were repeated three times and data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01 vs. corresponding control group. ADAM, disintegrin and metalloproteinase domain-containing protein; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Over Expression, Migration, Cell Counting, Transfection, Wound Healing Assay, Standard Deviation, Control, Small Interfering RNA, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Gene set enrichment analysis showed the enrichment plots of gene expression signatures for the Notch or Wnt signaling pathway according to ADAM17 expression. The barcode plot indicates the positions of genes in each gene set; red and blue colors indicate positive and negative correlations with ADAM17 expression, respectively. ADAM, disintegrin and metalloproteinase domain-containing protein; KEGG, Kyoto Encyclopedia of Genes and Genomes; Notch, neurogenic locus notch homolog protein.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Gene Expression, Expressing

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Results of gene set enrichment analysis showed key pathways correlated with ADAM17 expression in gastric cancer.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Expressing, Ubiquitin Proteomics, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

doi: 10.3892/ijmm.2018.4028

Figure Lengend Snippet: Western blot analysis of Notch2, GSK-3β, and β-catenin protein expression in SGC-7901 cells following suppression of ADAM17. All experiments were repeated three times and data are presented as the mean ± standard deviation. ** P<0.01, *** P<0.001 vs. the NC group. ADAM, disintegrin and metal-loproteinase domain-containing protein; Notch, neurogenic locus notch homolog protein; GSK-3β, glycogen synthase kinase-3β; siRNA, small interfering RNA; NC, negative control.

Article Snippet: Following blocking in 1X Tris-buffered saline/0.05% Tween-20 (TBST) supplemented with 5% non-fat milk at room temperature for 1 h, primary antibodies against ADAM17 (1:1,000; cat. no. 20259-1-AP; ProteinTech Group, Inc.), ADAM9 (1:1,000; cat. no. PA5-25959; Thermo Fisher Scientific, Inc.), neurogenic locus notch homolog protein (Notch)2 (1:1,000; cat. no. WL02409; Wanleibio Co., Ltd., Shanghai, China); glycogen synthase kinase (GSK)-3β (1:1,000; cat. no. 22104-1-AP; ProteinTech Group, Inc.), β-catenin (1:1,000; cat. no. 8480S; Cell Signaling Technology, Inc., Danvers, MA, USA) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) were added to the membranes and incubated overnight at 4°C. β-actin was used as the internal control.

Techniques: Western Blot, Expressing, Standard Deviation, Small Interfering RNA, Negative Control

Journal: PLoS Biology

Article Title: Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2

doi: 10.1371/journal.pbio.3000967

Figure Lengend Snippet: WT, TNFR1 -/- , and TNFR2 -/- mice were subjected to overload pressure for 2 weeks by TAC, and sham-operated mice served as controls. (A and C) Representative western blots of TNFR1, TNFR2, and tmTNF-α in myocardial tissues and quantitative data. (B) Quantitative RT-PCR analysis of TNF-α ( n = 5 per group). (D and G) Representative images of indirect fluorescence costaining for troponin T and tmTNF-α or TACE on myocardial sections (400×). (E and F) sTNF-α concentrations in heart homogenates and serum detected by ELISA ( n = 4 to 5 per group). (H–N) BALB/c mice were injected via the tail vein with rAAV-shTNFR1 or rAAV-shTNFR2 (1 × 10 11 virion particles). rAAV-GFP served as a control. After 2 weeks, the mice were subjected to sham operation or TAC for 14 days ( n = 6 per group). (H and K) Representative western blots for TNFR1, TNFR2, and tmTNF-α in myocardial tissues and quantitative data for tmTNF-α. (I and J) Quantitative RT-PCR analysis of ANP and BNP ( n = 5 per group). (L and M) Concentrations of sTNF-α in heart homogenates and serum determined by ELISA ( n = 5 per group). (N) Representative images of fluorescence immunostaining for TACE and troponin T in myocardial sections (400×). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sham. See individual data at and underlying raw images at . ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TAC, transverse aortic constriction; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane tumor necrosis factor-alpha; TNFR, TNF receptor; WT, wild-type.

Article Snippet: After stimulation, cells were washed with cold PBS and incubated with a polyclonal antibody against TNF-α (LifeSpan BioSciences, Seattle, Washington state, USA) or TACE (ProSci, San Diego, California, USA) for 1 h at 4°C, followed by an FITC-labeled secondary antibody against rabbit IgG (Jackson Biotech, West Chester, Pennsylvania, USA) ( ) for 1 h at 4°C.

Techniques: Western Blot, Quantitative RT-PCR, Fluorescence, Enzyme-linked Immunosorbent Assay, Injection, Control, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2

doi: 10.1371/journal.pbio.3000967

Figure Lengend Snippet: ISO (10 μM) was added to H9C2 cells for 24 h after a 24-h transfection with siRNA targeting TNFR1 or TNFR2 or to primary cardiomyocytes from WT, TNFR1-KO, or TNFR2-KO mice. DMSO served as a vehicle control. (A) Representative images of H9C2 cells stained with Actin-Trakcer Green (200×) and quantitative data of the cell surface area. Scale bar, 50 μm. Quantitative RT-PCR analysis of ANP (B and G), BNP (C and H), and TACE (F and K). (D and I) Representative cytograms and quantitative data for tmTNF-α expression in cardiomyocytes detected by flow cytometry . (E and J) sTNF-α levels in supernatants of H9C2 or primary cardiomyocytes determined by ELISA. All quantitative data represent the means ± SEs of at least 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 versus vehicle. Find individual data at . ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ISO, isoproterenol; KO, knockout; KD, knockdown; RT-PCR, real-time PCR; siRNA, small interfering RNA; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.

Article Snippet: After stimulation, cells were washed with cold PBS and incubated with a polyclonal antibody against TNF-α (LifeSpan BioSciences, Seattle, Washington state, USA) or TACE (ProSci, San Diego, California, USA) for 1 h at 4°C, followed by an FITC-labeled secondary antibody against rabbit IgG (Jackson Biotech, West Chester, Pennsylvania, USA) ( ) for 1 h at 4°C.

Techniques: Transfection, Control, Staining, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Peptide ELISA, Knock-Out, Knockdown, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: PLoS Biology

Article Title: Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2

doi: 10.1371/journal.pbio.3000967

Figure Lengend Snippet: TAPI-1 (8 mg/mL) was administered to WT mice by implantation of osmotic pumps (0.25 μL/h) directly after TAC or sham operation ( n = 6 per group). (A) Representative images of tmTNF-α fluorescence immunostaining on myocardial sections (400×) and quantitative data ( n = 5 per group). (B) Serum levels of sTNF-α detected by ELISA ( n = 3 to 4 per group). (C) Western blot analysis of tmTNF-α, TNFR1, and TNFR2. (D) Heart size and quantitative data of HW/BW ratio (mg/g). Scale bar, 3 mm. (E) Assessment of EF. (F–J), Quantitative RT-PCR analysis of ANP , BNP , IL-1β , IL-6 , and IL-10 in myocardial tissues ( n = 3 to 4 per group). ** P < 0.01, *** P < 0.001 versus corresponding group in sham. Individual data can be found in and underlying raw images in . ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HW/BW, heart weight to body weight; IL, interleukin; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TAC, transverse aortic constriction; TACE, TNF-α-converting enzyme; TAPI-1, TNF-α protease inhibitor-1; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.

Article Snippet: After stimulation, cells were washed with cold PBS and incubated with a polyclonal antibody against TNF-α (LifeSpan BioSciences, Seattle, Washington state, USA) or TACE (ProSci, San Diego, California, USA) for 1 h at 4°C, followed by an FITC-labeled secondary antibody against rabbit IgG (Jackson Biotech, West Chester, Pennsylvania, USA) ( ) for 1 h at 4°C.

Techniques: Fluorescence, Immunostaining, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Protease Inhibitor

Journal: PLoS Biology

Article Title: Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2

doi: 10.1371/journal.pbio.3000967

Figure Lengend Snippet: Primary cardiomyocytes from WT, TNFR1-KO, or TNFR2-KO mice were treated with ISO (10 μM) for 24 h in the presence or absence of exogenous sTNF-α (20 ng/mL) and tmTNF-α on fixed NIH3T3 cells (at an effector/target ratio of 10:1). Vector-transfected NIH3T3 cells served as a control. (A and C) Quantitative RT-PCR analysis of TACE . (B and D) Representative cytograms and quantitative data for TACE expression on the cell surface of cardiomyocytes detected by flow cytometry . All quantitative data represent means ± SEs of 5 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 versus corresponding control. See individual data at . FSC, Forward scatter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ISO, isoproterenol; KO, knockout; KD, knockdown; RT-PCR, real-time PCR; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor; WT, wild-type.

Article Snippet: After stimulation, cells were washed with cold PBS and incubated with a polyclonal antibody against TNF-α (LifeSpan BioSciences, Seattle, Washington state, USA) or TACE (ProSci, San Diego, California, USA) for 1 h at 4°C, followed by an FITC-labeled secondary antibody against rabbit IgG (Jackson Biotech, West Chester, Pennsylvania, USA) ( ) for 1 h at 4°C.

Techniques: Plasmid Preparation, Transfection, Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Knock-Out, Knockdown, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2

doi: 10.1371/journal.pbio.3000967

Figure Lengend Snippet: In contrast to sTNF-α that exerts prohypertrophy, and pro-inflammation through activating NF-κB pathway and inhibiting AKT pathway via TNFR1 (A), tmTNF-α displays antihypertrophy and anti-inflammation through suppressing NF-κB pathway and activating AKT pathway via TNFR2 in pressure overload–induced cardiac hypertrophy (B). In addition, mechanical stress induces TACE expression followed by enhanced release of sTNF-α that increases TACE expression via TNFR1, which cleaves tmTNF-α to produce more sTNF-α to display detrimental effects (A), whereas inhibition of TACE increases expression of tmTNF-α that down-regulates pressure overload–induced TACE expression via TNFR2, which in turn reduces tmTNF-α processing, and consequence increases tmTNF-α expression to display cardioprotective activities (B). IL, interleukin; NF-κB, nuclear factor-kappa B; sTNF-α, soluble TNF-α; TACE, TNF-α-converting enzyme; tmTNF-α, transmembrane TNF-α; TNFR, TNF receptor.

Article Snippet: After stimulation, cells were washed with cold PBS and incubated with a polyclonal antibody against TNF-α (LifeSpan BioSciences, Seattle, Washington state, USA) or TACE (ProSci, San Diego, California, USA) for 1 h at 4°C, followed by an FITC-labeled secondary antibody against rabbit IgG (Jackson Biotech, West Chester, Pennsylvania, USA) ( ) for 1 h at 4°C.

Techniques: Expressing, Inhibition