Journal: Cancer Science

Article Title: Inhibition of ADAM17 reduces hypoxia‐induced brain tumor cell invasiveness

doi: 10.1111/j.1349-7006.2007.00440.x

Figure Lengend Snippet: Effect of human a disintegrin and metalloproteinase (ADAM) 17 small interfering RNA (siRNA) transfection on ADAM17 mRNA expression and activity in U87 cells under normoxic and hypoxic conditions. ADAM17 mRNA and activity levels were significantly increased under hypoxic conditions compared to normoxic conditions. ADAM17 siRNA transfection reduced ADAM17 mRNA expression and activity in both normoxic and hypoxic conditions.

Article Snippet: Human TACE siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Small Interfering RNA, Transfection, Expressing, Activity Assay

Journal: Cancer Science

Article Title: Inhibition of ADAM17 reduces hypoxia‐induced brain tumor cell invasiveness

doi: 10.1111/j.1349-7006.2007.00440.x

Figure Lengend Snippet: Effect of human a disintegrin and metalloproteinase (ADAM) 17 small interfering RNA (siRNA) transfection on the in vitro invasiveness of U87 brain tumor cells cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. (a) Representative pictures of U87 cell invasion assay with different treatments. 1, Cells under normoxic conditions transfected with control siRNA; 2, cells under normoxic conditions transfected with ADAM17 siRNA; 3, cells under hypoxic conditions transfected with control siRNA; 4, cells under hypoxic conditions transfected with ADAM17 siRNA. (b) Relative invasive cell number compared to normoxic control. Data are shown as percentage of results under normoxic conditions. The values indicated by two asterisks (**) were significantly different (P < 0.01) from normoxia; the values indicated by hatches (# P < 0.01) were significantly different from the hypoxic control.

Article Snippet: Human TACE siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Small Interfering RNA, Transfection, In Vitro, Cell Culture, Invasion Assay, Control

Journal: Cancer Science

Article Title: Inhibition of ADAM17 reduces hypoxia‐induced brain tumor cell invasiveness

doi: 10.1111/j.1349-7006.2007.00440.x

Figure Lengend Snippet: Western blot analysis for a disintegrin and metalloproteinase (ADAM) 17, epidermal growth factor receptor (EGFR), phosphorylated EGFR (p‐EGFR), serine/threonine kinase (AKT) and phosphorylated AKT (p‐AKT) protein levels in U87 cells after small interfering RNA (siRNA) transfection. U87 cells transfected with siRNA cultured under normoxic and hypoxic conditions for 24 h. Protein levels of ADAM17, p‐EGFR, and p‐AKT were all significantly increased under hypoxic conditions; protein levels of EGFR and AKT were not significantly altered by hypoxia. ADAM17 siRNA transfection decreased ADAM17, p‐EGFR and p‐AKT protein levels in both normoxic and hypoxic conditions. EGFR and AKT protein levels were not significantly altered by ADAM17 siRNA in either normoxic or hypoxic conditions.

Article Snippet: Human TACE siRNA and control siRNA were purchased from Santa Cruz.

Techniques: Western Blot, Small Interfering RNA, Transfection, Cell Culture

Journal: Cell systems

Article Title: Protease Activity Profiling via Programmable Phage Display of Comprehensive Proteome-Scale Peptide Libraries.

doi: 10.1016/j.cels.2020.08.013

Figure Lengend Snippet: Figure 2. SEPARATE Detects Known Protease Targets at or below Physiological Concentrations (A) EpitopeFindR output as a network graph. Nodes represent caspase-1 digested peptides, and edges indicate regions of sequence homology. Multiple sequence alignment of the cluster containing the most peptides reveals a known caspase cleavage motif. (B) HUWE1 peptide tiles 51, 52, and 53 are cleaved by caspase-1. (C) Putative HUWE1 cleavage site identified by SEPARATE. (D) Western blot analysis of the time-dependent cleavage of HUWE1 in unstimulated THP-1 lysate after addition of recombinant caspase-1. (E) HUWE1 (top) and gasdermin D (GSDMD, bottom) are cleaved by endogenous caspase-1 in THP-1 cells upon inflammasome activation with indicated amounts of LPS and 2.5 mM Nigericin. (F) Aggregated data from a series of digests using thrombin, ADAM17, and caspase-1. Physiological concentrations for thrombin and ADAM17 are 1–1,000 and 1–10 nM; A range for capsase-1 is not reported. Cardiac troponin T (TNNT2; C) is a known thrombin substrate; Vasorin (VASN; A ) is a known substrate of ADAM17; and IL-1b (,) and HUWE1 (-) known substrates of caspase-1. (G–I) MA plot analysis of thrombin (G) (2 pM), (H) ADAM17 (0.4 nM), and (I) caspase-1 in THP1 lysate (0.3 mM).

Article Snippet: Protease digests were performed in 50 ml of appropriate buffer alone or containing the protease: (a) Caspase-1 (BioVision, Cat. No. 1081)/PreScission (GE Life Sciences, Cat. No. GE27-0843-01): TBS, pH 7.4, 0.01% NP40, 1 mM DTT; (b) Thrombin (SignalChem, Cat. No. T565-31N): TBS, pH 7.4, 0.01% NP40; (c) ADAM17 (RnD Systems, Cat. No. 930-ADB-010): Tris, pH 8.0, ZnCl2 2.5 uM, 0.005% Tween 20.

Techniques: Sequencing, Western Blot, Recombinant, Activation Assay

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