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

Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a, Reactome analysis of gene signatures in aberrant basal cells shows a strong inclination towards glucose-related metabolic pathways. b , Schematic representation of the cell isolation process from human distal lung tissue to generate patient-derived AOs. c , Representative immunofluorescence staining of 15 days AOs shows a pseudostratified epithelium with basal cells (KRT5+, green) externally, and differentiated club cells (SCGB1A1+, red) and goblet cells (MUC5B+, red) internally. Nuclei are stained with DAPI (blue). Scale bars are 20 µm (first row) and 50 µm (second row). d , Quantification of ciliated cell area indicates a significant decrease in cilia in IPF AOs from n = 6 IPF and n = 6 control AO donors (mean + s.e.m, * p < 0.05, unpaired t -test). e , Organoid size quantification over 15 days shows an increased proliferative capacity in IPF AOs (n = 4 IPF and n = 7 control AO donors, mean + s.e.m, **** p < 0.0001, unpaired t -test). f , Profibrotic biomarker MMP7 secretion is increased in IPF AOs (n = 5 IPF and n = 5 control AO donors, mean + s.e.m., *** p < 0.001, unpaired t -test). g , Representative immunofluorescence staining of AOs reveals aberrant basal cells (KRT5+(green)/KRT17+ (yellow)/COL1A1+ (red)). Nuclei are stained with DAPI (blue). Scale bars: 50 µm. IPF-stimuli further enriches this population after 7 days. h, i , Seahorse XF Mito Fuel Flex Test kit showed increased glucose ( h ) and decreased glutamine ( i ) dependency of IPF and IPF-stimulated AOs (n = 5 IPF and n = 5 control donors, mean + s.e.m., * p < 0.05, ** p < 0.01, ANOVA/ Tukey’s).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Cell Isolation, Derivative Assay, Immunofluorescence, Staining, Biomarker Assay

Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a , Representative western blot analysis and quantification (right panel) of total O-GlcNAc levels in control (n = 6) and IPF (n = 5) lung lysates revealed significant increase of O-GlcNAc in IPF lungs (violin plot, ** p < 0.01, unpaired t -test). b , c , RT-PCR analysis of MMP10 ( b ) and FN1 ( c ) in OGT-deleted airway epithelial cells treated with IPF-stimuli shows that OGT is required for profibrotic genes expression (n = 6, mean + s.e.m, * p < 0.05, **** p < 0.0001, ANOVA/Tukey’s). d, RT-PCR analysis of COL1A1 in IPF AOs treated with IPF-stimuli and OSMI-4 for 7 days shows decrease of gene expression upon OGT inhibition (n = 7, mean + s.e.m, ** p < 0.01, **** p < 0.0001, ANOVA/Tukey’s). e , ELISA analysis shows a decline in MMP7 secretion in an OGT-dependent manner upon stimulation with IPF-stimuli (n = 8, mean + s.e.m, * p < 0.05, ** p < 0.01, ANOVA/Friedman). f , RT-PCR analysis shows that OGT inhibition attenuates chronically injured epithelial-fibroblast coculture induced COL1A1 expression levels (n = 5, mean + s.e.m, ** p < 0.01, *** p < 0.001, ANOVA/Tukey’s). g , ELISA analysis reveals that OGT inhibition in chronically injured epithelial-fibroblast coculture ameliorates MMP7 secretion (n = 5, mean + s.e.m, * p < 0.05, ANOVA/Tukey’s). h, Quantification of ciliated cell area upon OSMI-4 treatment and chronic injury in epithelial-mesenchymal coculture increased area covered by ciliated cells (n = 3, mean + s.e.m, * p < 0.05, ** p < 0.01, ANOVA/Tukey’s). i , Quantification of average organoid size shows decreased organoid growth upon inhibition of OGT after 10 days (n = 5, mean + s.e.m, ** p < 0.01, ANOVA/Tukey’s). j , Representative immunofluorescence staining of IPF and control AOs treated with IPF-stimuli and OSMI-4 for 7 days shows decrease in aberrant basal cell signature upon OGT inhibition (scale bar 50 µm).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: JUNB O-GlcNAcylation-mediated promoter accessibility of metabolic genes modulates distinct epithelial lineage in pulmonary fibrosis

doi: 10.1101/2024.05.27.594700

Figure Lengend Snippet: a , Representative western blot analysis of the O-GlcNAc immunoprecipitated fraction shows increased levels of O-GlcNAc mark on JUNB in injured airway epithelial cells (n = 3 independent donors). IgG antibody was employed as negative control. b , c , RT-PCR analysis shows diminished expression of pro-fibrotic genes ( COL1A1 ( b ) and MMP10 ( c )) upon transfection of JUNB-LOF (loss-of-function) and fibrotic induction IPF-stimuli in airway epithelial cells (n = 5, mean + s.e.m, * p < 0.05, ** p < 0.01, **** p < 0.0001, ANOVA/Tukey’s) Plasmid was generated by site-specific mutations in the O-GlcNAc sites of JUNB. d , Transduction of JUNB-LOF plasmid in n = 6 IPF AOs led to a decrease in the secretion of MMP7 after 10 days (mean + s.e.m, * p < 0.05, Wilcoxon). e , f , Representative pictures ( e ) and quantification ( f ) of average organoids size of n = 6 IPF AOs was reduced after JUNB-LOF transduction in AOs after 10 days, whereas overexpression of JUNB led to a significant increase in growth (mean + s.e.m, * p < 0.05, ANOVA/Tukey’s).

Article Snippet: Measurement of human MMP7 (Biotechne, #DY907) was performed according to manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Generated, Transduction, Over Expression