Journal: Biomedicines

Article Title: Consequences of the Lack of TNFR1 in Ouabain Response in the Hippocampus of C57BL/6J Mice

doi: 10.3390/biomedicines10112937

Figure Lengend Snippet: Effect of ouabain and the lack of TNFR1 in TNF levels, TNFR2 expression, and TACE/ADAM17 activity. ( A ) TNF levels in serum were not changed by ouabain treatment or the TNFR1 gene. ( B ) TNFR1 KO mice presented the same TNF levels as WT mice in hippocampus. ( C ) Densitometric analysis and representative Western blotting of TNFR2 membrane expression. There was an increase in TNFR2 expression in the membrane enriched fraction for the gene factor [F (1, 30) = 5.591]. ( D ) The activity of TACE/ADAM 17 was raised for ouabain treatment [F (1, 16) = 4563]. Results are expressed in pg/mL for serum samples ( n = 11, N = 3) and pg/υg (mean ± SEM) in hippocampal samples ( n = 4, N = 2) for TNF measurements, in control ratio for TNFR2 expression ( n = 5, N = 2), and ng/mg in TACE/ADAM 17 activity ( n = 5, N = 1). Two-way ANOVA was performed for the comparison followed by Tukey´s post-test.

Article Snippet: A mouse TACE/ADAM17 kit was used to measure ADAM17 activity (BT Lab).

Techniques: Expressing, Activity Assay, Western Blot, Membrane, Control, Comparison

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

Journal: International Journal of Hypertension

Article Title: miR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding

doi: 10.1155/2023/9497716

Figure Lengend Snippet: Expression of miR-145, ADAM17, ACE2, and Mas receptor in the aortic arteries of hypertensive rats. 22 male Wistar rats were selected randomly to receive standard diet or high-sucrose/high-fat diet for 30 weeks, the thoracic aorta was collected, the expression of ACE2 was detected by Western blotting, and the expression of MASR, miR-145, and ADAM17 was detected by qPCR. (a) Quantification of ACE2 expression from panel (b) data was expressed after normalization to the β -actin. (b) Representative figures from Western blotting of ACE2. (c–e) Quantification of mRNA expression of MASR, miR-145, and ADAM17 (qPCR). ∗∗ P < 0.01 between groups and ∗ P < 0.05 between groups.

Article Snippet: Antibodies against angiotensin converting enzyme 2 (ACE2) (21115-1-AP), Mas receptor (20080-1-AP), osteopontin (OPN) (22952-1-AP), α -SMA (Proteintech, USA, 1 : 2000), SM22a (10493-1-AP), ADAM17 (20259-1-AP), EREG (CSB-PA189260), MMP2 (10373-2-AP) and GAPDH (10494-1-AP), and HRP goat anti-rabbit IgG (SA00001-2) were purchased from Proteintech (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Hypertension

Article Title: miR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding

doi: 10.1155/2023/9497716

Figure Lengend Snippet: miR-145 augments Ang II-induced ACE2-Ang-(1–7)-Mas axis activation and ADAM17 expression in VSMCs. VSMCs were treated with control, Ang II (1 μ M), miR-145 mimic (100 nM), or miR-145 (100 nM) inhibitor alone or in combination for 48 hours. (a) Concentration of Ang-(1–7) in the supernatant (ELISA). (b, c) ACE2 and MASR expression from panel (e) and data are expressed as the fold of the GAPDH. (d) ADAM17 expression in groups from panel (f). (e, f) Representative figures of ACE2, MASR, and ADAM17 expression in VSMCs (Western blotting). ∗∗ P < 0.01 vs. control group; ∗ P < 0.05 vs. control group; ## P < 0.01 vs. Ang II group; and # P < 0.05 vs. Ang II group. All the data are expressed as mean ± SEM from three independent experiments. miRNA NC, negative control miRNA.

Article Snippet: Antibodies against angiotensin converting enzyme 2 (ACE2) (21115-1-AP), Mas receptor (20080-1-AP), osteopontin (OPN) (22952-1-AP), α -SMA (Proteintech, USA, 1 : 2000), SM22a (10493-1-AP), ADAM17 (20259-1-AP), EREG (CSB-PA189260), MMP2 (10373-2-AP) and GAPDH (10494-1-AP), and HRP goat anti-rabbit IgG (SA00001-2) were purchased from Proteintech (Proteintech, USA).

Techniques: Activation Assay, Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control

Journal: International Journal of Hypertension

Article Title: miR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding

doi: 10.1155/2023/9497716

Figure Lengend Snippet: ADAM17 siRNA reversed phenotype transition induced by miR-145 in vitro. VSMCs were treated with miR-145 inhibitor in the presence or absence of ADAM17 siRNA (100 nM) as indicated for 48 hours. The expression of α -SMA (a), SM22 α (b), OPN (c), EREG (d), and MMP2 (e) protein was detected by Western blotting. All the data were normalized to that of GAPDH. (f) Representative figures of OPN, α -SMA, SM22 α , EREG, and MMP2 (Western blotting). (g) The luciferase reporter assay is shown. Cells were transfected with a reporter vector psiCHECK-2-ADAM17 3′-UTR plus either miR-145-5p or the negative control. ∗∗ P < 0.01 vs. control group or miR-145-5p NC and ## P < 0.01 vs. miR-145 inhibitor group. All the data are expressed as mean ± SEM of three independent experiments. NC siRNA, negative control siRNA. WT, wide type. MT, mutant type.

Article Snippet: Antibodies against angiotensin converting enzyme 2 (ACE2) (21115-1-AP), Mas receptor (20080-1-AP), osteopontin (OPN) (22952-1-AP), α -SMA (Proteintech, USA, 1 : 2000), SM22a (10493-1-AP), ADAM17 (20259-1-AP), EREG (CSB-PA189260), MMP2 (10373-2-AP) and GAPDH (10494-1-AP), and HRP goat anti-rabbit IgG (SA00001-2) were purchased from Proteintech (Proteintech, USA).

Techniques: In Vitro, Expressing, Western Blot, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Negative Control, Control, Mutagenesis

Journal: International Journal of Hypertension

Article Title: miR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding

doi: 10.1155/2023/9497716

Figure Lengend Snippet: ADAM17 mediated miR-145-induced effect by regulating ACE2-Ang-(1–7)-Mas axis in vitro. VSMCs were treated with miR-145 inhibitor in the presence or absence of ADAM17 siRNA (100 nM) as indicated for 48 hours. (a) Concentration of Ang-(1–7) in the supernatant (ELISA). (b) Representative figures of ACE2 and MASR (Western blotting). Quantification of ACE2 (c) and MASR (d) expression determined by Western blotting, and the data were normalized to that of GAPDH. ∗∗ P < 0.01 vs. control group; ∗ P < 0.05 vs. control group; and ## P < 0.01 vs. miR-145 inhibitor group. All the data are expressed as mean ± SEM of three independent experiments. NC siRNA, negative control siRNA.

Article Snippet: Antibodies against angiotensin converting enzyme 2 (ACE2) (21115-1-AP), Mas receptor (20080-1-AP), osteopontin (OPN) (22952-1-AP), α -SMA (Proteintech, USA, 1 : 2000), SM22a (10493-1-AP), ADAM17 (20259-1-AP), EREG (CSB-PA189260), MMP2 (10373-2-AP) and GAPDH (10494-1-AP), and HRP goat anti-rabbit IgG (SA00001-2) were purchased from Proteintech (Proteintech, USA).

Techniques: In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Negative Control

Journal: Brain

Article Title: Cerebrovascular lesions induce transient ?-amyloid deposition

doi: 10.1093/brain/awr300

Figure Lengend Snippet: Transient middle cerebral artery occlusion did not affect proteins related to generation or degradation of β-amyloid. Representative examples of the limited effect of middle cerebral artery occlusion on proteins related to β-amyloid production or degradation using immunohistochemistry in post-mortem tissue sections. BACE, presenilin 1 and APP immunoreactivity was observed in the surrounding of senile plaques (not labelled), whereas neprilysin and insulin degrading enzyme signal was detected in neurons. No differences were observed in BACE (A and B), presenilin 1 (C and D), APP (E and F), neprilysin (G and H) or insulin degrading enzyme (I and J) (top: ipsilateral hemisphere; bottom: contralateral hemisphere). Scale bar = 200 μm.

Article Snippet: Reagents Texas Red® dextran of 70 000 D molecular weight was obtained from Molecular Probes; methoxy-XO4 was a gift from Dr Klunk (University of Pittsburgh); Ketamine HCl and xylazine were obtained from Phoenix Pharmaceuticals, anti-β-amyloid antibody (10D5) (developed in mouse) was a gift from Elan Pharmaceuticals; anti-β-amyloid-converting enzyme (BACE) antibody (developed in rabbit) was obtained from ProSci Incorporated; anti-amyloid precursor protein (APP) antibody (developed in rabbit) was obtained from Calbiochem; anti-insulin degrading enzyme (developed in goat) was obtained from Santa Cruz; and anti-neprilysin (developed in rat) was obtained from R&D Systems.

Techniques: Immunohistochemistry

Journal: Frontiers in bioscience (Landmark edition)

Article Title: MiR-145 Alleviates Sepsis-Induced Inflammatory Responses and Organ Injury by Targeting ADAM17.

doi: 10.31083/j.fbl2901044

Figure Lengend Snippet: Fig. 1. Lipopolysaccharide (LPS) reduces miR-145 expression, overexpression of miR-145 downregulated ADAM17 expression

Article Snippet: The membranes were incubated with antibodies against ADAM17 (sc-390859, mouse monoclonal antibody, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA; A00604, rabbit polyclonal antibody, 1:800, Boster Biological Technology, China) or β-actin (sc-47778, mouse monoclonal antibody, 1:4000, Santa Cruz Biotechnology, USA) at 4 °C overnight.

Techniques: Expressing, Over Expression

Journal: Frontiers in bioscience (Landmark edition)

Article Title: MiR-145 Alleviates Sepsis-Induced Inflammatory Responses and Organ Injury by Targeting ADAM17.

doi: 10.31083/j.fbl2901044

Figure Lengend Snippet: Fig. 2. miR-145 alleviated LPS-induced endothelial inflammation by targeting ADAM17 in HUVECs. HUVECs were infected by LV-NC, LV-miR-145, LV-ADAM17-siRNA or the combined of LV-miR-145 and LV-ADAM17-siRNA. (A) Green fluorescence protein

Article Snippet: The membranes were incubated with antibodies against ADAM17 (sc-390859, mouse monoclonal antibody, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA; A00604, rabbit polyclonal antibody, 1:800, Boster Biological Technology, China) or β-actin (sc-47778, mouse monoclonal antibody, 1:4000, Santa Cruz Biotechnology, USA) at 4 °C overnight.

Techniques: Infection, Fluorescence

Journal: Frontiers in bioscience (Landmark edition)

Article Title: MiR-145 Alleviates Sepsis-Induced Inflammatory Responses and Organ Injury by Targeting ADAM17.

doi: 10.31083/j.fbl2901044

Figure Lengend Snippet: Fig. 3. Overexpression of miR-145 reduces expression of ADAM17, attenuates sepsis-induced inflammatory responses and acute lung injury. (A) Schematic of experimental design and time line. Polymicrobial sepsis model of mice was induced in C57BL/6 mice

Article Snippet: The membranes were incubated with antibodies against ADAM17 (sc-390859, mouse monoclonal antibody, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA; A00604, rabbit polyclonal antibody, 1:800, Boster Biological Technology, China) or β-actin (sc-47778, mouse monoclonal antibody, 1:4000, Santa Cruz Biotechnology, USA) at 4 °C overnight.

Techniques: Over Expression, Expressing

Journal: Frontiers in bioscience (Landmark edition)

Article Title: MiR-145 Alleviates Sepsis-Induced Inflammatory Responses and Organ Injury by Targeting ADAM17.

doi: 10.31083/j.fbl2901044

Figure Lengend Snippet: Fig. 4. MiR-145 agomir reduces expression of ADAM17, attenuates sepsis-induced acute kidney injury and offers survival benefit.

Article Snippet: The membranes were incubated with antibodies against ADAM17 (sc-390859, mouse monoclonal antibody, 1:800, Santa Cruz Biotechnology, Santa Cruz, CA, USA; A00604, rabbit polyclonal antibody, 1:800, Boster Biological Technology, China) or β-actin (sc-47778, mouse monoclonal antibody, 1:4000, Santa Cruz Biotechnology, USA) at 4 °C overnight.

Techniques: Expressing