Journal: PLoS ONE

Article Title: Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

doi: 10.1371/journal.pone.0091596

Figure Lengend Snippet: (A) The mRNA expression levels of uPA, Cathepsin B, MMP-2 and different EMMPRIN isoforms in head and neck cancer cells were examined after exogenous expression of EMMPRIN-2. Transfection of an EMMPRIN-2 expression vector or siRNA (siEMMPRIN) selectively enhanced or attenuated the mRNA expression levels of EMMPRIN-2 in both ACCM and Tca8113 cells, while the expression of other EMMPRIN isoforms, uPA, Cathepsin B and MMP-2 remained unaffected ( p >0.05). (B) The mRNA expression findings were confirmed by analyzing protein expression using western blot. (C) Detection of extracellular MMP-2, Cathepsin B and uPA using ELISA. MMP-2, Cathepsin B and uPA secretion into the culture medium was increased in cells that had been transfected with the EMMPRIN-2 expression vector, while secretion was decreased in EMMPRIN-2 siRNA transfected cells ( p <0.05). (D) Analysis of extracellular MMP-2 activity using a gelatin zymography assay. Extracellular MMP-2 activity was increased after EMMPRIN-2 expression was enhanced, while MMP-2 activity was reduced after EMMPRIN-2 expression knockdown.

Article Snippet: Next, the cells were incubated with 15 ml of serum-free media, and the media was collected using an Amicon Ultra 15 ml 10K column (Millipore) after 24 hours. uPA activity, MMP-2 and Cathepsin B concentrations were quantified using a uPA Activity Assay Kit (Millipore), a Human MMP-2 Quantikine ELISA Kit (R&D Systems) and a Human Cathepsin B Duo Set Kit (R&D Systems) according to the manufacturers recommended protocols.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Zymography Assay

Journal: PLoS ONE

Article Title: Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

doi: 10.1371/journal.pone.0091596

Figure Lengend Snippet: (A) Treatment with Cathepsin B siRNA resulted in corresponding reductions in Cathepsin B mRNA levels in both EMMPRIN-2 overexpressing and parental Tca8113 cells ( p <0.05), while EMMPRIN-2 and MMP-2 were unaffected by Cathepsin B siRNA transfection ( p >0.05). (B) The effects on Cathepsin B, EMMPRIN-2 and MMP-2 expression following Cathepsin B siRNA transfection in EMMPRIN-2 overexpressing and parental Tca8113 cells were further confirmed using western blot analyses. (C) The extracellular secretion of Cathepsin B was also reduced by siRNA treatment in Tca8113 cells both in the presence and absence of EMMPRIN-2 overexpression ( p <0.05). (D) As observed in the experiments above, Tca8113 cells overexpressing EMMPRIN-2 displayed increased cell invasion, migration and adhesion as compared to parental Tca8113 cells. In contrast, down-regulation of Cathepsin B using siRNA significantly inhibited the invasion, migration and adhesion of Tca8113 cells overexpressing EMMPRIN-2.

Article Snippet: Next, the cells were incubated with 15 ml of serum-free media, and the media was collected using an Amicon Ultra 15 ml 10K column (Millipore) after 24 hours. uPA activity, MMP-2 and Cathepsin B concentrations were quantified using a uPA Activity Assay Kit (Millipore), a Human MMP-2 Quantikine ELISA Kit (R&D Systems) and a Human Cathepsin B Duo Set Kit (R&D Systems) according to the manufacturers recommended protocols.

Techniques: Transfection, Expressing, Western Blot, Over Expression, Migration

Journal: Journal of Ophthalmology

Article Title: Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF- κ B Pathway in Cultured Human Uveal Melanoma Cells

doi: 10.1155/2016/8734309

Figure Lengend Snippet: Zeaxanthin inhibits secretion of MMP-2 protein by cultured UM cells. UM cells were seeded into 12-well plates and were cultured with or without zeaxanthin (10 μ M). After being cultured for 24 h, conditioned medium was collected and centrifuged and the supernatants were collected. The amount of MMP-2 in the supernatants was determined using the Human MMP-2 ELISA kit. Zeaxanthin at 3 μ M and 10 μ M significantly inhibited the secretion of MMP-2 ( n = 3, P < 0.05). ∗ P < 0.05, versus control (cells cultured without zeaxanthin).

Article Snippet: The protein amount of MMP-2 in the conditioned media was determined using the Human MMP-2 Quantitation ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncology Letters

Article Title: Effect of epithelial growth factor on matrix metalloproteinase-2 and E-cadherin/β-catenin expression in an in situ model of tumorigenesis

doi: 10.3892/ol.2017.6513

Figure Lengend Snippet: MMP-2 expression levels without EGF (control) or with 5 and 10 ng/ml EGF. The values are representative of three independent experiments conducted in duplicate (mean ± standard deviation). *P<0.05, compared with the control group. MMP-2, matrix metalloproteinase-2; EGF, epidermal growth factor.

Article Snippet: ELISA MMP-2 quantification was performed using the human MMP-2 ELISA kit (catalog no. DY902; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Standard Deviation