Journal: PLoS ONE

Article Title: Exendin-4 Induces Cell Adhesion and Differentiation and Counteracts the Invasive Potential of Human Neuroblastoma Cells

doi: 10.1371/journal.pone.0071716

Figure Lengend Snippet: Representative experiment from three independent experiments on the invasive ability of SK-N-AS and SH-SY5Y cells after treatment with 0.3 µM exendin-4 (A); real-time RT-PCR analysis of the expression of MMP-9 and TIMP-1 in NB cells after treatment with 0.3 µM exendin-4 for 6 h. * = p<0.05 vs. related control (B). Size of the cell colonies grown in soft agar 7, 14 or 21 days after suspension, with or without (C = control) exendin-4. Data are reported as mean percentage vs. related control of three replicates. EXE = exendin-4; * = p<0.05 vs. related control; # = p<0.05 vs. C 7d; § = p<0.05 vs. C 14d (C).

Article Snippet: Primers and probe for uPAR (Hs00182181_m1), CXCR4 (Hs02330069_s1), microtubule-associated protein-2 MAP2 (Hs00159041_m1), Tau (Hs00902193_m1), Synaptophisin (Hs00300531_m1), Tissue inhibitor of metalloproteinases-1 (TIMP-1) (Hs99999139_m1), Matrix Metalloproteinase-9 (MMP-9) (Hs00957562_m1) were Assay-On-Demand products (Applied Biosystem).

Techniques: Quantitative RT-PCR, Expressing, Control, Suspension

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 2 Loci-specific transactivation of the mouse TIMP promoters using dCas9.nVP64. Notes: Fold-induction over the no gRNA transfection controls using dCas9.nVP64 (black circle) or dCas9 (orange triangle) and the relative gRNAs targeting loci within the (A) TIMP1, (B) TIMP2, or (C) TIMP3 core promoters. Closed symbols represent targeting of the top strand (also indicated by T) while open symbols represent targeting of the bottom strand (indicated by B) of the promoter DNA. X-axis shows the relative distance from the start codon and the numerous transcription factor-binding sites located within the mouse TIMP promoters. Data from Clark et al.24 Mean±standard error of the mean , n=3. Abbreviation: TIMP, tissue inhibitor of metalloproteinases.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Transfection, Binding Assay

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 3 Comparison of the transactivation efficiency of TIMP1 and TIMP2 promoters using different transcriptional activating effectors. Notes: (A) Illustration of the different dCas9 constructs used. (B, C) Fold luciferase induction over no gRNA transfection controls comparing VP64-, VP160-, or VPR. dCas9 effector constructs targeting 3 loci within the mouse TIMP1 (B) or 4 loci within the mouse TIMP2 (C) promoter. Mean±standard error of the mean (SEM), *P<0.025, **P<0.005, ***P<0.0005, ****P<0.0001, unpaired t-test, n=4. (D, E) Fold luciferase induction over no gRNA transfection comparing VPR targeting 4 loci in the mouse TIMP1 (D) and mouse TIMP2 (E) promoter to SAM targeting between 0 and 250 bps upstream of the transcription start site. Mean±SEM, **P<0.01, ***P<0.005, one-way analysis of variance with multiple comparisons, n=4. Abbreviations: CMV, cytomegolovirus; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; NLS, nuclear localization sequence; SAM, synthetic activation motif; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Comparison, Construct, Luciferase, Transfection, Sequencing, Activation Assay, Virus

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 4 Endogenous TIMP gene activation in mouse NSC34 cells. Notes: (A) PMA stimulation enhances promoter-reporter activity for TIMP1 and 3. TIMP promoter-reporter constructs were transfected into HEK293 cells. The promoter activity (luciferase) was assessed by the Dual-Glo assay 12 hours after DMSO or PMA stimulation at the indicated concentrations. Mean±standard error of the mean (SEM), *P<0.05, **P<0.01 or n.s., n=3. (B) Endogenous TIMP mRNA basal expression in unstimulated NSC34 cells relative to GAPDH. Mean±SEM, n=6–11. All bars were significantly different (P<0,001) from each other by a pair-wise two-tailed t-test. Specific endogenous Cas9-VPR mediated transactivation using gRNAs targeting the mouse TIMP1 (C) and mouse TIMP2 (D) promoters. Dashed bar indicates the expression of all 4 gRNAs simultaneously. Mean±SEM, *P<0.05, **P<0.01, one-way analysis of variance with multiple comparisons, n=4. The “All” condition was analyzed separately; mean±SEM, ****P<0.0001, unpaired t-test, n=4. Abbreviations: DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; n.s., not significant; PMA, phorbol-12-myristate-13-acetate; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Activation Assay, Activity Assay, Construct, Transfection, Luciferase, Glo Assay, Expressing, Two Tailed Test

Journal: Metalloproteinases In Medicine

Article Title: dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

doi: 10.2147/mnm.s146752

Figure Lengend Snippet: Figure 5 Production of functional TIMP2 protein by gRNA-induced NSC34 cells. Notes: (A) Reverse zymography of cell culture supernatant. The lanes were loaded with control recombinant TIMP1 or 2 (10, 3,1, or 0 ng), or cell culture supernatant from NSC34 cells transfected with nothing, dCas-VPR alone, dCas- VPR with a negative control blank gRNA, or dCas-VPR with all 4 (p-194, p-535p, p-610, and p-956) TIMP2 gRNAs. The transfection condition was identical to that for mRNA assay except that the culture supernatant was harvested 48 hours after transfection. The blot is representative of 3 biological replicates. (B) Time course of increase in fluorescence due to cleavage of the fluorogenic MMP substrate. The slope of the fluorescence vs time plot was taken as a measure of the relative catalytic activity. The plot is representative of 3 biological replicate experiments. (C) A bar plot of relative MMP9 catalytic activity (mean±standard error of the mean, n=3). Note significant inhibition (****P<0.0001) of the catalytic activity by cell culture supernatant from wells transfected with all 4 TIMP2 gRNAs. Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Article Snippet: The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Techniques: Functional Assay, Zymography, Cell Culture, Control, Recombinant, Transfection, Negative Control, Fluorescence, Activity Assay, Inhibition

Journal: International Journal of Medical Sciences

Article Title: Comparison the Prognostic Value of Galectin-3 and Serum Markers of Cardiac Extracellular Matrix Turnover in Patients with Chronic Systolic Heart Failure

doi: 10.7150/ijms.8083

Figure Lengend Snippet: Receiver operating characteristic (ROC) curves for prediction mortality. The area under the curve (AUC) of log MMP-2, log TIMP-1, log BNP, log PIIINP, log Gal-3, log PINP, and log MMP-9 were 0.786, 0.699, 0.636, 0.625, 0.607, 0.519, and 0.367, respectively. Abbreviations: BNP= brain natriuretic peptide; Gal-3= Galectin-3; HF=heart failure; LVEF= left ventricular ejection fraction; MMP = matrix metalloproteinase; PINP = type I amioterminal propeptide of procollagen; PIIINP = type III amioterminal propeptide of procollagen; TIMP = tissue inhibitor of metalloproteinase.

Article Snippet: TIMP-1 was measured by an ELISA kit (DTM100, R & D systems, Minneapolis, USA).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Comparison the Prognostic Value of Galectin-3 and Serum Markers of Cardiac Extracellular Matrix Turnover in Patients with Chronic Systolic Heart Failure

doi: 10.7150/ijms.8083

Figure Lengend Snippet: Kaplan-Meier analysis of cumulative rates of survival in HF patients with higher or lower levels of serum Gal-3 or cardiac ECM markers. The p value of Gal-3, PINP, PIIINP, TIMP-1, MMP-2, MMP-9, and BNP were 0.153, 0.708, 0.154, 0.028, 0.001, 0.501, and 0.483, respectively. Abbreviations: BNP= brain natriuretic peptide; ECM= Extracellular matrix; Gal-3= Galectin-3; HF=heart failure; LVEF= left ventricular ejection fraction; MMP = matrix metalloproteinase; PINP = type I amioterminal propeptide of procollagen; PIIINP = type III amioterminal propeptide of procollagen; TIMP = tissue inhibitor of metalloproteinase.

Article Snippet: TIMP-1 was measured by an ELISA kit (DTM100, R & D systems, Minneapolis, USA).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Comparison the Prognostic Value of Galectin-3 and Serum Markers of Cardiac Extracellular Matrix Turnover in Patients with Chronic Systolic Heart Failure

doi: 10.7150/ijms.8083

Figure Lengend Snippet: Kaplan-Meier analysis of cumulative rates of HF admission-free survival in HF patients with higher or lower levels of serum Gal-3 or cardiac ECM markers. The p value of Gal-3, PINP, PIIINP, TIMP-1, MMP-2, MMP-9, and BNP were 0.166, 0.624, 0.639, 0.085, <0.001, 0.624, and 0.684, respectively. Abbreviations: BNP= brain natriuretic peptide; ECM= Extracellular matrix; Gal-3= Galectin-3; HF=heart failure; MMP = matrix metalloproteinase; PINP = type I amioterminal propeptide of procollagen; PIIINP = type III amioterminal propeptide of procollagen; TIMP = tissue inhibitor of metalloproteinase.

Article Snippet: TIMP-1 was measured by an ELISA kit (DTM100, R & D systems, Minneapolis, USA).

Techniques:

Journal: Biomedical Reports

Article Title: Anti-allergic effect of the naturally-occurring conjugated linolenic acid isomer, jacaric acid, on the activated human mast cell line-1

doi: 10.3892/br.2015.517

Figure Lengend Snippet: Jacaric acid modulates the expression levels of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) proteins in human mast cell line-1 (HMC-1) cells. HMC-1 cells were incubated with 1 (lane 2), 2 (lane 3) and 4 µM jacaric acid (lane 4) at 37°C for 72 h. Cells treated with 0.02% ethanol (lane 1) acted as the control. Subsequently, treated cells were further incubated with Iono and phorbol 12-myristate 13-acetate at 37°C for 6 h. (A) Protein expression levels of MMP-2, MMP-9 and TIMP-1 were assayed by western blotting with β-actin protein as an internal control. (B-D) The relative expression levels of MMP-9, MMP-2 and TIMP-1 proteins compared to β-actin were quantified. Results represent mean ± standard error. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: The membrane was first incubated with the following primary antibodies: Rabbit anti-MMP-2 (CST-4022S), anti-MMP-9 (CST-3852S), anti-tissue inhibitor of metalloproteinase-1 (TIMP-1) (CST-8946S) (Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse anti-β-actin antibody (SC-A5316) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), followed by incubation with mouse IgG horseradish peroxidase-conjugated secondary antibody (GE-NA931) or rabbit IgG horseradish peroxidase-conjugated secondary antibody (GE-NA934) (GE Healthcare, Buckinghamshire, UK and finally developed with the enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Incubation, Control, Western Blot