Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: Recombinant MMP7 and galectin-3 were incubated together for various time points. Reaction products were separated on SDS-PAGE, transferred to PVDF membranes and proteins detected by Coommassie Brilliant Blue staining. A. A 4 hr reaction mixture revealed three possible galectin-3 cleavage products (Bands A, B and C). B. A time course revealed galectin-3 cleavage within 5 minutes with the early appearance of Bands B and C. Band A became the predominant MMP7-induced galectin-3 cleavage product at the 24 hr incubation time point.

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Recombinant, Incubation, SDS Page, Staining

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: A. Mass spectra of recombinant galectin-3 and MMP7 reaction products. Recombinant MMP7 and galectin-3 were incubated together for 4 and 24 hours and mass spectrometry performed. B. Coomassie Brilliant Blue staining of the 4 and 24 hr reaction mixtures utilized for subsequent N-terminal sequencing. C. Schematic representation of MMP7-directed galectin-3 cleavage sites for the 20.2, 18.9 and 15.5 kDa galectin-3 fragments. (figure not to scale.)

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Recombinant, Incubation, Mass Spectrometry, Staining, Sequencing

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: Recombinant MMP7 and galectin-3 were incubated together for 1 hr and cleavage reaction products were detected with an anti-galectin-3 C-terminus antibody (A.) and an anti-galectin-3 N-terminus antibody (B.) MMP7-induced galectin-3 cleavage products were only detected with the anti-galectin-3 C-terminus antibody.

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Recombinant, Incubation

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: A. T84 cell colonies were stained for anti-galectin-3 C-terminus (red), MMP7 (green) and DAPI (blue) and visualized by confocal microscopy. Predominant MMP7 and galectin-3 staining were seen in the periphery with co-localization (yellow) seen in the merged image. B. Western blot analysis detected the 28 kDa pro-MMP7 in T84 cell lysates and culture medium supernatants. C. Western blot analysis detected the full length galectin-3 protein in the T84 culture medium supernatants.

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Staining, Confocal Microscopy, Western Blot

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: T84 cells were incubated with (50 and 100 nM) MMP7 for 4 hours and presence of galectin-3 in culture supernatants detected by Western blot analysis. The 15.5 and 20.2 kDa galectin-3 cleavage products were detected in MMP7-treated cells while only the full length galectin-3 was detected in untreated T84 cell culture supernatants.

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques: Incubation, Western Blot, Cell Culture

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: Confluent T84 cell monolayers were wounded and immediately treated with: galectin-3, galectin-3 + β-lactose, anti-galectin-3 neutralizing antibody, active MMP7, anti-MMP7 neutralizing antibody, and active MMP7 + anti-MMP7 neutralizing antibody. A. Photomicrographs were obtained at 0 and 24 hours. B. Quantitative analysis of wound width at 0 and 24 hrs was performed and percent wound width covered calculated (n = 6 per condition; * p < 0.01).

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques:

Journal:

Article Title: Matrilysin-1 (MMP7) cleaves galectin-3 and inhibits wound healing in intestinal epithelial cells

doi: 10.1002/ibd.21443

Figure Lengend Snippet: Confluent T84 cell monolayers were wounded in the presence of galectin-3, MMP7 and galectin-3 plus MMP7. Quantitative analysis of wound width at 0 and 24 hrs was performed and percent wound width covered calculated (n = 6 per condition; * p < 0.01).

Article Snippet: The mouse anti-MMP7 monoclonal antibody was obtained from R&D Systems (Minneapolis, MN, USA) and goat anti-GAPDH polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Techniques:

Journal: Molecular cancer research : MCR

Article Title: Cooperativity of E-cadherin and Smad4 Loss to Promote Diffuse-type Gastric Adenocarcinoma and Metastasis

doi: 10.1158/1541-7786.MCR-14-0192-T

Figure Lengend Snippet: (A) Representative immunohistochemistry findings for E-cadherin and β-catenin at the invasive front of tumors across genotypes. (B) Percentage of nuclear β-catenin-postive cells at the invasive front of gastric adenocarcinomas from Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ cohort (n=14) and Pdx-1-Cre;Trp53F/F;Cdh1F/F cohort (n=4) and duodenal carcinomas from Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice (n=4). Box indicates interquartile range with median. (C) Vimentin and MMP7 immunohistochemistry at the invasive front of gastric adenocarcinomas between Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ and Pdx-1-Cre;Trp53F/F;Cdh1F/F mice. (D) Percentage of Vimentin- and MMP7-positive cells in gastric adenocarcinomas arising in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice (n=10) and Pdx-1-Cre;Trp53F/F;Cdh1F/F mice (n=5).

Article Snippet: The following antibodies were used: rabbit polyclonal anti-E-cadherin antibody (1:200; Cell Signaling, #3195, Danvers, MA), rabbit polyclonal anti-Ki-67 antibody (1:200; Abcam, ab15580, Cambridge, UK), mouse monoclonal anti- β -catenin antibody (1:200; BD Transduction Laboratories, 610154, San Diego, CA), rabbit monoclonal anti-vimentin antibody (1:100; Cell Signaling, #5741), and rabbit monoclonal anti-MMP7 antibody (1:100; Cell Signaling, #3801).

Techniques: Immunohistochemistry

Journal: Molecular cancer research : MCR

Article Title: Cooperativity of E-cadherin and Smad4 Loss to Promote Diffuse-type Gastric Adenocarcinoma and Metastasis

doi: 10.1158/1541-7786.MCR-14-0192-T

Figure Lengend Snippet: Inhibition of β-catenin by Smad4/BMP pathway. (A and B) Ctnnb1 mRNA expression in primary cultured cells according to Smad4 status. (A) Real-time RT-PCR for Ctnnb1 mRNA in primary cultured gastric adenocarcinomas cells from Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ and Pdx-1-Cre;Trp53F/F;Cdh1F/F mice. Expression levels of Ctnnb1 after treatment with 100 ng/ml of BMP2, 400 ng/ml of noggin (a BMP antagonist) and 5 ng/ml of TGF-β1 for 16 hours. (Bi) Real-time RT-PCR for Ctnnb1 mRNA in Smad4 overexpressing stable transfectants of Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ primary cells (Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+-Smad4) vs. vector only-transfected Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ primary cells (Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+-Mock). (Bii) Smad4 Western blot analyses on (Bi). (C and D) β-catenin reporter activity in (A and B). Tcf/Lef reporter activity was lower in Smad4 expressing cell lines (Pdx-1-Cre;Trp53F/F;Cdh1F/F and Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+-Smad4) than in Smad4-null cells. (E) Migratory activity of primary cultured cells from gastric adenocarcinomas arising from Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ (n=4) and Pdx-1-Cre;Trp53F/F;Cdh1F/F mice (n=2). (F) Migratory activity of two independent primary cultured cell lines from gastric adenocarcinomas arising from Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice, after knockdown of β-catenin using two different short hairpin RNAs. Western blot analysis showed the efficiency of β-catenin knockdown. (G) Representative image for shCtnnb1-1-tranfected Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ cell line 2 (spindle-like shape) is shown with scrambled control-transfected cells (epithelial, cuboidal shape). (H) β-catenin knockdown-induced changes in mRNA expression of Cdh2, MMP7 and EMT-activating transcription factors in (G). Asterisks (*) denote the statistical significance (P<0.05). Bars indicate standard errors of the mean. Representative data for repeated experiments are shown.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-E-cadherin antibody (1:200; Cell Signaling, #3195, Danvers, MA), rabbit polyclonal anti-Ki-67 antibody (1:200; Abcam, ab15580, Cambridge, UK), mouse monoclonal anti- β -catenin antibody (1:200; BD Transduction Laboratories, 610154, San Diego, CA), rabbit monoclonal anti-vimentin antibody (1:100; Cell Signaling, #5741), and rabbit monoclonal anti-MMP7 antibody (1:100; Cell Signaling, #3801).

Techniques: Inhibition, Expressing, Cell Culture, Quantitative RT-PCR, Plasmid Preparation, Transfection, Western Blot, Activity Assay, Knockdown, Control

Journal: Cellular Physiology and Biochemistry

Article Title: Cyclic Stretch Magnitude and Duration Affect Rat Alveolar Epithelial Gene Expression

doi:

Figure Lengend Snippet: Top: MMP7 Cassiene Zymogram demonstrating reduction the white regions associated with enzyme activity. The lower band is 18 kDa active MMP7 in the supernatant. Each 100 μg sample was pooled from the 6-8 wells of the same isolation. Bottom: The quantization of the active MMP7 intensities (N=3 isolation/group) values. Black bar is control, dotted bar is 25*1, and dashed bar is 25*6.

Article Snippet: Aliquots (2 μl) of the cDNA (Rn00567471_m1, Rn00563101_m1, Rn00563467_m1, Rn00576196_m1, Rn00695641_m1, and Rn99999916_s1, respectively, from Applied Biosystems) were used in TaqMan gene expression assays to detect encoding in mRNAs obtained for each of the following experimental groups: 25*1, 25*6, and unstretched controls (N=3 rats/group), and mRNA levels were quantified in triplicate according to the supplier's recommendations.

Techniques: Activity Assay, Isolation, Control