mmp 15 overexpression  (OriGene)

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