Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: List of the analyzed ECM components.

Article Snippet: MMP-2 , TA806846 , OriGene Technologies (Rockville, MD, USA) , 1:200 , Human tonsil.

Techniques:

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Primary antibodies used for immunohistochemical staining.

Article Snippet: MMP-2 , TA806846 , OriGene Technologies (Rockville, MD, USA) , 1:200 , Human tonsil.

Techniques: Immunohistochemical staining, Staining, Positive Control

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Invasion spectrum (the mRNA expression pattern of invasion-associated extracellular matrix components) differs in patients with ‘worse’ and ‘better’ prognoses. mRNA expression measurements were performed twice for each gene to confirm the data. A longer bar on the logarithmic scale indicates reduced expression. *P<0.05 vs. group A (Mann-Whitney U test). Group A, OS <24 months; group B, OS >24 months; BCAN, brevican; CD44, cluster of differentiation 44; CSPG5, chondroitin sulfate proteoglycan 5; EGFR, epidermal growth factor receptor; FLT4, Fms-related tyrosine kinase 4; HMMR, hyaluronan-mediated motility receptor; IDH1, isocitrate dehydrogenase 1; ITGAV, integrin-αV; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; NCAN, neurocan; PDGFA, platelet-derived growth factor α; TNC, tenascin C; VCAN, versican; OS, overall survival.

Article Snippet: MMP-2 , TA806846 , OriGene Technologies (Rockville, MD, USA) , 1:200 , Human tonsil.

Techniques: Expressing, MANN-WHITNEY, Derivative Assay

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: A total of 3 invasion-associated extracellular matrix components demonstrated significantly different expression levels in the samples from different prognostic groups. All 3 molecules had increased expression in patients in group A, indicating that the level of these molecules was associated with tumor invasiveness and patient survival. Group A, OS <24 months; group B, OS >24 month.; FLT4, Fms-related tyrosine kinase 4; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; rel., relative; OS, overall survival.

Article Snippet: MMP-2 , TA806846 , OriGene Technologies (Rockville, MD, USA) , 1:200 , Human tonsil.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Immunohistochemical images of glioblastoma stained for MMP-2. (A) MMP-2 positivity was notably strong in immunohistochemical images from patients with a worse prognosis, confirming the mRNA expression results. (B) Patients with a better prognosis expressed an increased amount of MMP-2, as the strong positivity indicates, but the immunohistochemical scores are significantly reduced. (C) Negative control slide. All images are depicted at ×20 magnification. MMP-2, matrix metallopeptidase 2.

Article Snippet: MMP-2 , TA806846 , OriGene Technologies (Rockville, MD, USA) , 1:200 , Human tonsil.

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling

doi: 10.1152/ajpcell.00137.2016

Figure Lengend Snippet: Oligonucleotides for quantitative PCR

Article Snippet: Antibodies used for Western blotting are as follows: MMP2 (catalog no. AF1488, R & D Systems), MMP9 (catalog no. AF909, R & D Systems), MMP13 (catalog no. sc-30073, Santa Cruz Biotechnology), β-catenin (catalog no. 9582, Cell Signaling Technology), collagen type 1 (catalog no. sc-28657, Santa Cruz Biotechnology), SMA (catalog no. ab5694, Abcam), and YY1 ( 19 ).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling

doi: 10.1152/ajpcell.00137.2016

Figure Lengend Snippet: sFRP2 stimulates expression of matrix metalloproteinase (MMP) 1a and MMP13 in CFs. CFs (A and B) and C2C12 myoblasts (C) were transfected with pcDNA3 (Control) and pCMV-sFRP2 vector DNA. In A and C, total RNA was harvested from cells after 3 days and processed for quantitative PCR analysis of MMP gene expression. *P < 0.05 vs. Control. Only MMP1a and MMP13 are shown for C2C12 myoblasts. In B, conditioned media were analyzed by Western blotting using MMP13 and MMP2 antibodies. MMP protein abundance was quantified by densitometry, and MMP13 protein levels are presented as MMP13-to-MMP2 ratio.

Article Snippet: Antibodies used for Western blotting are as follows: MMP2 (catalog no. AF1488, R & D Systems), MMP9 (catalog no. AF909, R & D Systems), MMP13 (catalog no. sc-30073, Santa Cruz Biotechnology), β-catenin (catalog no. 9582, Cell Signaling Technology), collagen type 1 (catalog no. sc-28657, Santa Cruz Biotechnology), SMA (catalog no. ab5694, Abcam), and YY1 ( 19 ).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Quantitative Proteomics

Journal: American Journal of Physiology - Cell Physiology

Article Title: sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling

doi: 10.1152/ajpcell.00137.2016

Figure Lengend Snippet: sFRP2 enhances enzyme activities of MMP2 and MMP9 in CFs. Conditioned media were harvested from CFs (A and C), as well as C2C12 myoblasts and liver fibroblasts (B), 3 days after DNA transfection. Intensity of proMMP and mature MMP forms was measured by gelatin zymography (20 μl of medium loaded per lane) followed by densitometry. Quantification is presented for CF media in A. *P < 0.05 vs. Control. In C, sFRP2-treated conditioned medium was incubated with 1 mM 4-aminophenylmercuric acetate (APMA) for 1 h prior to gelatin zymography. Respective gel positions of proMMP and active MMP forms are indicated.

Article Snippet: Antibodies used for Western blotting are as follows: MMP2 (catalog no. AF1488, R & D Systems), MMP9 (catalog no. AF909, R & D Systems), MMP13 (catalog no. sc-30073, Santa Cruz Biotechnology), β-catenin (catalog no. 9582, Cell Signaling Technology), collagen type 1 (catalog no. sc-28657, Santa Cruz Biotechnology), SMA (catalog no. ab5694, Abcam), and YY1 ( 19 ).

Techniques: Transfection, Zymography, Control, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling

doi: 10.1152/ajpcell.00137.2016

Figure Lengend Snippet: Downregulation of collagen type 1 by sFRP2 in CF conditioned medium. A: total RNA was harvested from CFs after 3 days and processed for quantitative PCR analysis of expression of Col1A1 and Col1A2 genes. B–D: conditioned media harvested from CFs, C2C12 myoblasts, and liver fibroblasts 3 days after DNA transfection were processed for Western blot analysis of procollagen 1 (30 μl of medium loaded per lane). Densitometric quantification of proCol1A1 intensity is presented as collagen-to-MMP2 ratio. *P < 0.05 vs. Control.

Article Snippet: Antibodies used for Western blotting are as follows: MMP2 (catalog no. AF1488, R & D Systems), MMP9 (catalog no. AF909, R & D Systems), MMP13 (catalog no. sc-30073, Santa Cruz Biotechnology), β-catenin (catalog no. 9582, Cell Signaling Technology), collagen type 1 (catalog no. sc-28657, Santa Cruz Biotechnology), SMA (catalog no. ab5694, Abcam), and YY1 ( 19 ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: sFRP2 activates Wnt/β-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling

doi: 10.1152/ajpcell.00137.2016

Figure Lengend Snippet: sFRP2-Wnt signaling regulation of MMPs and collagen turnover. CFs were treated with BIO (5 μM), IWR-1 (3 μM), or DMSO (Control) upon completion of the 6-h transfection protocol. Conditioned media were collected and analyzed by gelatin zymography (A and B). C: quantification of data from B. D: Western blotting of procollagen 1. MMP protein intensity was quantified for the sFRP2 treatment group and is presented as proCol1A1-to-MMP2 ratio. *P < 0.05 vs. Control.

Article Snippet: Antibodies used for Western blotting are as follows: MMP2 (catalog no. AF1488, R & D Systems), MMP9 (catalog no. AF909, R & D Systems), MMP13 (catalog no. sc-30073, Santa Cruz Biotechnology), β-catenin (catalog no. 9582, Cell Signaling Technology), collagen type 1 (catalog no. sc-28657, Santa Cruz Biotechnology), SMA (catalog no. ab5694, Abcam), and YY1 ( 19 ).

Techniques: Control, Transfection, Zymography, Western Blot

Journal: Cancer Cell International

Article Title: Aurora-B knockdown inhibits osteosarcoma metastasis by inducing autophagy via the mTOR/ULK1 pathway

doi: 10.1186/s12935-020-01674-1

Figure Lengend Snippet: Aurora-B knockdown-induced autophagy inhibits migration and invasion in OS cells. The migration and invasion ability of Aurora-B-knockdown 143B and HOS cells treated with or without CQ (12 μM, 12 h) were examined by transwell migration ( a , b *** P < 0.01, Scale bars: 100 μm) and invasion ( c , d * P < 0.05, **** P < 0.01, *** P < 0.01,** P < 0.01, Scale bars: 100 μm) assays in vitro. Wound healing assay was performed with scrambled and shAurora-B 143B and HOS cells treated with CQ (12 μM, 12 h) for 24 h or not ( e , f ** P < 0.01, *** P < 0.01, * P < 0.05, Scale bars: 100 μm). MMP2 protein expression of Aurora-B-knockdown cells treated with CQ (10 μM, 24 h) or not was analyzed by western blotting ( g )

Article Snippet: The primary antibodies included rabbit anti-human AuroraB (Abcam ab45145), anti-β-tubulin (Abcam ab179513), anti-SQSTM1/P62 (Cell signaling technology 5114), rabbit anti-LC3B (Cell signaling technology 2775), anti-p-mTOR(ser2448)(Cell signaling technology 2971), anti-mTOR(Cell signaling technology 2972), anti-AMPK (Cell Signaling Technology, 2532), anti-pAMPKα (Thr172) (Cell Signaling Technology, 2535), anti-ULK1 (Cell Signaling Technology, 8054) and anti-Pulk1 (Ser555) (Cell Signaling Technology, 5869), mouse anti-human GAPDH (Origene TA802519) and anti-MMP2 (Origene TA806846S).

Techniques: Migration, In Vitro, Wound Healing Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Relative mRNA levels of TCF-4 and MMP-15 in SAEC, H460, A549 and LLC cells (n = 5, **P < 0.01). ( b ) Relative mRNA levels of TCF-4 and MMP-15 in the lung tissues or LLC-tumor tissues. The 8-week old female C57BL/6 mice were intravenously injected with LLC cells (5 × 10 5 cells in 100 μl PBS). Two weeks later, the inoculated LLC-tumors or adjacent lung tissues were collected for mRNA assays (n = 5, **P < 0.01). ( c , d ) Relative mRNA levels of TCF-4 ( c ) and MMP-15 ( d ) in the lung carcinoma or adjacent normal tissues from human subjects (n = 10, **P < 0.01). The experiment in ( a , b ) was performed in triplicate. The experiment in ( c , d ) was repeated twice.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Injection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a , b ) The mRNA ( a ) and protein ( b ) levels of TCF-4 and MMP-15 in SAEC cells which were transfected with PCMV (0.4 μg/ml) or PCMV-TCF-4 (0.4 μg/ml) for 24 h (n = 5, **P < 0.01). ( c , d ) The mRNA ( c ) and protein ( d ) levels of TCF-4 and MMP-15 in LLC cells which were transfected with a scramble siRNA (si-NC, 20 nmol/ml) or the mouse TCF-4 specific siRNAs (si-TCF4-1 or si-TCF4-2, 20 nmol/ml) for 36 hours (n = 3, *P < 0.05). The experiment from ( a – d ) was repeated in triplicate and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter gene (m-P1) (0.4 μg/ml) harboring the mouse MMP-15 promoter sequences (-3000/-1) for 36 hours (n = 5, **P < 0.01). ( b ) Relative luciferase activity of SAEC cells transfected with si-NC (20 nmol/ml) or a siRNA for human TCF-4 (si-TCF4, 20 nmol/ml) plus the reporter gene (h-P1) (0.4 μg/ml) harboring the human MMP-15 promoter sequences (-3000/-1) for 36 hours (n = 5, **P < 0.01). ( c ) A schematic depiction of different mouse MMP-15 promoter regions which were cloned into the pGL4-basic plasmid. The constructs were designated as m-P1~m-P3 as indicated. ( d ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes m-P1~m-P3 (0.4 μg/ml) for 36 hours (n = 5, **P < 0.01). The tests in ( a , b , d ) were repeated in triplicate.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Luciferase, Activity Assay, Transfection, Clone Assay, Plasmid Preparation, Construct

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) The conserved promoter region and NF-κB binding sites between human and mouse MMP-15 gene. The human MMP-15 promoter sequences (-3000/-1) were aligned with the mouse MMP-15 promoter sequences (-3000/-1). Two conserved NF-κB binding elements in the mouse MMP-15 promoter (-2958/-2949, -2833/-2824) and human MMP-15 promoter (-2456/-2447, -2348/-2339) were mutated and constructed as reporter genes m-MUT1, m-MUT2, h-MUT1 and h-MUT2, respectively as indicated. ( b ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes m-P1, m-MUT1 and m-MUT2 (0.4 μg/ml) for 36 hours, respectively (n = 5, **P < 0.01). ( c ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus the reporter genes h-P1, h-MUT1 and h-MUT2 (0.4 μg/ml) for 36 hours, respectively (n=5, **P < 0.01). The experiment in ( b , c ) was repeated for 3 times.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Binding Assay, Construct, Luciferase, Activity Assay, Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) TCF-4 potentiates the binding between NF-κB p65 and the MMP-15 promoter. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by p65 antibody or rabbit IgG as control. ( b ) TCF-4 interacts with NF-κB p65 which binds to the promoter of MMP-15. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for ChIP assay. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( c ) Protein-protein interaction between TCF-4 and NF-κB p65 in LLC cells. The LLC cells were transfected with si-NC (20 nmol/ml), si-TCF4-1 (20 nmol/ml) or si-TCF4-2 (20 nmol/ml) for 36 hours, and then harvested for immunoprecipitation (IP) and Western blotting assay of p65 protein. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. ( d ) Protein-protein interaction between TCF-4 and NF-κB p65 in SAEC cells. The SAEC cells were transfected with PCMV (0.4 μg/ml) or PCMV-TCF4 (0.4 μg/ml) for 36 hours, and then collected for IP and Western blotting assay of p65. The cell lysates were immunoprecipitated by TCF-4 antibody or rabbit IgG as control. Experiment from ( a – d ) was repeated for 3 times, and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with PCMV (0.4 μg/ml) or PCMV-TCF-4 (0.4 μg/ml) plus si-NC (20 nmol/ml) or a siRNA for mouse NF-κB p65 (si-p65, 20 nmol/ml) for 36 hours (n = 5, **P < 0.01). ( b ) The mRNA levels of MMP-15, TCF-4 and p65 in the LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus pCDNA3.1 (0.4 μg/ml) or pCDNA-p65 (0.4 μg/ml) for 36 hours (n = 5, **P < 0.01). ( c ) Immunoblotting assay of TCF-4 and p65 in the cytosol or nucleus of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) for 36 hours. ( d ) Relative luciferase activity of LLC cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus a reporter construct (0.4 μg/ml) containing the promoter of CCL20 for 36 hours (n = 4, **P < 0.01). The tests from ( a – d ) were repeated for 3 times, and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Construct

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) TCF-4 promotes LLC cell migration via MMP-15. The scratch tests were performed on the LLC cells which were transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml). The representative pictures were taken immediately after scratch (0 hours) or after a subsequent 18 hours (18 h). ( b ) Relative migration rate of the cells described in ( a ) (n = 5, **P < 0.01). ( c ) TCF-4 promotes MMP-15-dependent LLC cell migration. Transwell assays were carried out on the cells transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml). ( d ) Relative migrated cells described in ( c ) were counted (n = 5, **P < 0.01). The tests in ( a , c ) were repeated for 3 times and the representative images were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Migration, Transfection

Journal: Scientific Reports

Article Title: T cell factor-4 functions as a co-activator to promote NF-κB-dependent MMP-15 expression in lung carcinoma cells

doi: 10.1038/srep24025

Figure Lengend Snippet: ( a ) Representative images of LLC-tumors in the lung tissues. The LLC cells were transfected with si-NC (20 nmol/ml) or si-TCF4-1 (20 nmol/ml) plus PCMV (0.4 μg/ml) or PCMV-MMP-15 (0.4 μg/ml) for 24 hours. Then, the 8-week old female C57BL/6 mice were intravenously injected with those different LLC cells (5 × 10 5 cells in 100 μl PBS) as indicated. Two weeks later, the lung tissues were collected for pathological observation with H&E staining. ( b ) Relative tumor lesions displayed in ( a ) were calculated (n = 6, **P < 0.01). ( c ) Survival time of different LLC cells-inoculated mice. The 8-week old female C57BL/6 mice were intravenously injected with different LLC cells (5 × 10 6 cells in 100 μl PBS) as indicated. The survival time after tumor inoculation was recorded (n = 10, **P < 0.01). The experiment in ( a , c ) was repeated twice and the representative results were displayed.

Article Snippet: MMP-15 overexpression was performed by transfecting the cells with a commercial construct (MC202385, OriGene).

Techniques: Transfection, Injection, Staining