Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Western Blot, Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Over Expression, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Activity Assay, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: In Silico, Clinical Proteomics, Membrane, In Vitro, Recombinant, Western Blot, Cell Culture

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Flow Cytometry, Staining, Fluorescence, Two Tailed Test, Comparison

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Ex Vivo, In Vivo, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

doi: 10.1101/2020.04.23.057109

Figure Lengend Snippet: Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for an hour at 37°C. The reaction was stopped by the addition of KLK14-specific inhibitors and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 (A) negative control was not activated. ProMMP14 (B) , proMMP15 (C) and proMMP16 (D) were activated whereas proMMP17 (E) did not show hydrolysis of gelatin, yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems)).

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D systems), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D systems), 0.5 μg ProMMP16 (catalog no. 1785-MP-010, R&D systems) and 1 μg ProMMP17 (catalog no. 7796-MP-010, R&D systems) were separately incubated in 10 μl with a range of KLK14 concentrations (25-250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma) for 1 hour at 37°C in PBS.

Techniques: Activation Assay, Functional Assay, Incubation, SDS Page, Negative Control

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

Journal: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously [ ]. si GENOME SMART pool ® reagents were products of Dharmacon (Lafayette, CO).

Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: A plasmid encoding human MT2-MMP (TC118648) was purchased from Origene (Rockville, MD).

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation