mouse anti mmp14  (R&D Systems)

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: Expression of MMP14 during EMT of chick NC cells. (A) In situ hybridization against chick MMP14. Dorsal view of the caudal part of a stage HH13 chick embryo. Scale bar: 100 µm. (B-D) In situ hybridization for chick MMP14. Transverse sections. MMP14 expression is increased in the neural crest during EMT. Scale bars: 50 µm. s, somite; L, lateral mesoderm; np, neural plate; nt, neural tube; n, notochord. Asterisks indicate the pre-migratory NC cells.

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Expressing, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: MMP14 is required for NC delamination independently of its catalytic activity. (A-D) Immunostaining for snail 2 (magenta) after electroporation of scrambled control (A), siRNA-MMP14 (B) and co-electroporation of siRNA-MMP14 with wild-type MMP14 (C) or the non-catalytic point mutant MMP14-EA (D). Nuclei are counterstained with DAPI (grey); the electroporated side is visualized using GFP (green). Brackets indicate neural crest accumulation (B). Scale bars: 20 µm. (E) The method for measuring the area of the neural crest domain. The neural crest area on the electroporated side was divided by the neural crest area in the control side. (F) Plot of neural crest area (snail 2) with scrambled (nembryos=11, nsections=132 from three independent experiments), siRNA-MMP14 alone (nembryos=31, nsections=445 from eight independent experiments), siRNA-MMP14 with wild-type MMP14 (nembryos=7, nsections=134 from two independent experiments) and siRNA-MMP14 with MMP14-EA (nembryos=4, nsections=90 from one experiment). ANOVA with a Kruskal–Wallis Test and Dunn's multiple comparisons: ****P<0.0001 (scrambled versus siRNA), *P=0.0441 (siRNA versus siRNA+MMP14), **P=0.0030 (siRNA versus siRNA+EA) and P>0.9999 (not significant; scrambled versus siRNA+MMP14 or siRNA+EA).

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Activity Assay, Immunostaining, Electroporation, Control, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: MMP14 inhibition prevents the apicobasal redistribution of PCM1 during NC EMT. (A,B) immunostaining against aPKC (magenta) and snail 2 (gray) in embryos electroporated with scrambled control (A, nembryos=3) and with siRNA-MMP14 (B, nembryos=5) from one experiment. (C-D′) Immunostaining against PCM1 (magenta) and AP2 (gray) in embryos electroporated with scrambled control (C, nembryos=6) and with siRNA-MMP14 (D,D′, nembryos=5) from two independent experiments. (D′) More detail of the neural crest domain. Electroporated side is visualized using GFP (green). Bracket in D indicates the accumulation of neural crest cells. Arrowheads in C indicate the normal basolateral distribution of PCM1 on both non-electroporated and electroporated sides of embryos electroporated with the scrambled control. Arrowheads in D,D′ indicate the normal basolateral distribution of PCM1 on the control side of siRNA-MMP14 embryos. Arrows in D,D′ indicate the apical position of PCM1 on the electroporated side of siRNA-MMP14 embryos. Scale bars: 20 µm.

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Inhibition, Immunostaining, Control

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: MMP14 is required for the occurrence of basal mitoses during NC delamination. (A,B) Immunostaining for phospho-histone 3 (pH3) (yellow) and snail 2 (magenta) in electroporated embryos with scrambled control (A) and siRNA-MMP14 (B). GFP is shown in green, nuclei are counterstained with DAPI (gray). Arrowhead in A indicates an example of basal mitosis in the electroporated side. Arrow in B indicates an example of apical mitosis in the electroporated side. Scale bars: 20 µm. (C) Diagram depicting the method of measuring the positions of mitoses along the apical-basal axis. The distance between the mitoses and the apical part of neural tube (d) was divided by the neural tube width (D). A ratio of 0 means that the mitoses were close to the apical region. A ratio of 1 means that the mitoses were close to the basal region. (D) Plot of the position of the mitoses along the apico-basal axis in the neural crest in embryos electroporated with the scrambled control (nembryos=3, nmitoses non-elec side=33, nmitoses elec side=36 from one experiment) or siRNA-MMP14 (nembryos=8, nmitoses non-elec side=51, nmitoses elec side=69, from three independent experiments). Dashed line indicates the border between the apical half and basal half. ANOVA followed by Dunn's multiple comparisons **P=0.0014 (electroporated scrambled versus electroporated siRNA), **P=0.0027 (non-electroporated siRNA versus electroporated siRNA). In the scatter dot plot, the lines indicate the median and the error bars show the extent of the whole dataset. (E) Percentage of basal mitoses in neural crest domain. Basal mitoses were defined as mitoses located within the basal half of the neural crest domain (corresponding to values between 0.5 and 1 on the graph in D). Statistics were performed according to Taillard et al. (2008), **T>6.635 (non-electroporated siRNA versus electroporated siRNA) the null hypothesis is rejected with 99% confidence. No-elec, non-electroporated side; elec, electroporated side.

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Immunostaining, Control

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: Homogenous distribution of MMP14 is sufficient to promote basal mitoses independently of its catalytic activity. (A) Immunostaining against phospho-histone H3 (pH3, magenta) after electroporation with the scrambled control or siRNA-MMP14. (B) Positions of mitoses in the intermediate neural tube region with the scrambled control (nembryos=6, nmitoses=339; from two independent experiments) or siRNA-MMP14 (nembryos=11, nmitoses=362; from four independent experiments). Unpaired Mann–Whitney test, P=0.7780. (C) Diagram depicting the different forms of MMP14. (D) Immunostaining against pH3 (magenta) after overexpression of MMP14, MMP14-ΔCAT, MMP14-EA and MMP14-ΔCyto. Arrows indicate non-apical mitoses. (E) Positions of all mitoses for each condition depicted in D. Control side (nembryos=26, nmitoses=1908), MMP14 (nembryos=7, nmitoses=716), MMP14-ΔCAT (nembryos=7, nmitoses=406) and MMP14-EA (nembryos=9, nmitoses=707) from three independent experiments, and MMP14-ΔCyto (nembryos=3, nmitoses=431) from one experiment. (F) Percentages of non-apical mitoses for each condition depicted in D. Non-apical mitoses were determined as mitoses occurring more than two nuclei diameters away from the apical domain. Statistics were performed according to Taillard et al. (2008), Thresholds for T were such that, at *T>3.841, the null hypothesis is rejected with a 95% confidence, at **T>6.635, the null hypothesis is rejected with 99% confidence and at ***T>10.83, the null hypothesis is rejected with 99.9% confidence. In B,E, the lines indicate the median and the error bars show the extent of the whole dataset. Electroporated side is indicated by GFP (A,D, green). Scale bars: 35 µm.

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Activity Assay, Immunostaining, Electroporation, Control, MANN-WHITNEY, Over Expression

Journal: Development (Cambridge, England)

Article Title: MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity

doi: 10.1242/dev.183954

Figure Lengend Snippet: Destabilizing apicobasal polarity is sufficient to rescue siRNA-MMP14. (A) Immunostaining for snail 2 (gray) after electroporation of siRNA-MMP14 or co-electroporation with siRNA-MMP14 and Par3. Bracket indicates accumulation of neural crest cells. (B) Plot of neural crest area (snail 2) with siRNA-MMP14 (nembryos=14, nsections=361), siRNA-MMP14 with Par3 (nembryos=6, nsections=166) and siRNA-MMP14 with Par3MO (nembryos=7, nsections=38) from two independent experiments. ANOVA and uncorrected Fisher's LSD; ***P=0.0007, ****P<0.0001. Scale bars: 25 µm. Par3MO efficiency/specificity could not be checked (see main text and Materials and Methods for details).

Article Snippet: Western blot antibodies used were: custom-made polyclonal mouse anti-chick MMP14 (1/2500), rabbit anti-tubulin (Cell Signaling, 2148, 1/1000), anti-mouse PAR3 and anti-human Par3 (Millipore 07-330 and 8E8, respectively, 1/1000; clone 8E8 did not recognize chick-Par3, 07-330 produced multiple non-specific bands on top of the expected 180, 150 and 100 kDa bands, rendering the blots inconclusive), mouse anti-GFP (Roche, 11814460001, 1/1000) and rabbit anti-GFP (Merck, G1544, 1/1000).

Techniques: Immunostaining, Electroporation

Journal: Scientific Reports

Article Title: PROX1 is a transcriptional regulator of MMP14

doi: 10.1038/s41598-018-27739-w

Figure Lengend Snippet: PROX1 and MMP14 expressions are inversely correlated. ( a ) Representative image of a KS section from an AIDS patient stained with MMP14 (green) and Prox1 (magenta) specific antibodies. Nuclei were counterstained with Hoechst 33342. ( b ) MMP14 and Prox1 signal intensities for each cell were quantified from 3 images/section of 10 different patients. Each dot represents a cell and the different colours represent the different patients. ( c ) 57 specimens from TMA were stained as in ( a ), representative images are shown for one benign hyperplasia (left) and one papillary thyroid cancer (right). White arrows indicate cells with Prox1 cytoplasmic staining. ( d ) Transcript levels of Prox1 and MMP14 in different tissues/organs derived from FANTOM5 database. Tags per million for MMP14 (Blue) and Prox1(red) are shown for each tissue/organ. Pearson´s correlation coefficient (r): −0.4515. Significance of the correlation p = 0.0065.

Article Snippet: For the staining of mouse ears the following antibodies were used: rat anti-mouse PECAM1 (BD-Biosciences; 553370), rabbit anti-mouse LYVE-1 (103-PA50AG, Reliatech), goat anti-PROX1 (R&D AF2727), rat anti mouse endomucin (Santa Cruz sc-65495), hamster anti mouse podoplanin (Developmental Studies hybridoma bank 8.1.1) and mouse monoclonal anti-MMP14 (LEM clone; Millipore).

Techniques: Staining, Derivative Assay

Journal: Scientific Reports

Article Title: PROX1 is a transcriptional regulator of MMP14

doi: 10.1038/s41598-018-27739-w

Figure Lengend Snippet: PROX1 binds to the MMP14 promoter and regulates its expression. ( a ) Upper panel: schematic diagram of the MMP14 promoter fragments, numbers indicate the bp upstream (−) or downstream (+) of the MMP14 transcription start site (TSS, where bp = 0 is MMP14 TSS), indicated by the black arrow. Bottom panel: luciferase reporter assay using either the pGL3 backbone or its indicated derivatives depicted above. HeLa cells were co- transfected with the indicated reporter plasmids (0.1 µg) and expression vectors for PROX1 WT or MUT (1 µg) in duplicates. An empty pAMC vector was used as a control (mock). 32 h after transfection, cell extracts were collected and luciferase activities were measured. The graph includes the data from three independent experiments. Error bars represent SD. (b) Upper panel: schematic representation of the MMP14 promoter region spanning from bp −1340 to −36. Letters denote the fragments amplified by different sets of primers (a–h) used in the ChIP-qPCR below. Bottom panel: Chromatin immunoprecipitation using either PROX1 or control IgG antibodies followed by qPCR with primers amplifying the indicated regions of the 1.2 kb MMP14 promoter region. Average fold enrichment over the IgG is shown for three independent experiments; error bars represent SD. ( c ) Left: schematic representation of the 1.2 kb MMP14 promoter reporter plasmid, the b and c regions immunoprecipitated in ( b ) are marked as red boxes. PROX1 binding site containing regions (BS1 and BS2) are illustrated below as red dotted lines, numbers indicate their position upstream of the MMP14 TSS. These sequences were deleted in the 1.2 kb MMP14 promoter reporter plasmid (white boxes), generating the 1.2 kb MMP14 promoter mutants ΔBS1, ΔBS2, ΔBS1-2. Right: Luciferase assay performed as in ( a ) using 1.2 kb MMP14 promoter either full length (FL) or the indicated mutants co-transfected with either a pAMC control vector (mock) or a PROX1 WT expression vector. *p < 0.05; **p > 0.01; ***p > 0.001.

Article Snippet: For the staining of mouse ears the following antibodies were used: rat anti-mouse PECAM1 (BD-Biosciences; 553370), rabbit anti-mouse LYVE-1 (103-PA50AG, Reliatech), goat anti-PROX1 (R&D AF2727), rat anti mouse endomucin (Santa Cruz sc-65495), hamster anti mouse podoplanin (Developmental Studies hybridoma bank 8.1.1) and mouse monoclonal anti-MMP14 (LEM clone; Millipore).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay

Journal: Scientific Reports

Article Title: PROX1 is a transcriptional regulator of MMP14

doi: 10.1038/s41598-018-27739-w

Figure Lengend Snippet: PROX1 depletion increases MMP14 expression in vivo and in vitro . ( a ) Whole-mount immunofluorescence of 4 weeks old Prox1 flox ; Cdh5-CreER T2 mice ear skin. Note efficient depletion of PROX1 in the Prox1 flox/flox ; Cdh5-CreER T2 ear compared to the control. ( b ) Representative images of IHC staining of the ear sections from mice described in ( a ) for podoplanin and endomucin. Nuclei were counterstained with Hoechst 33342. ( c ) Representative images of IHC staining for MMP14 and LYVE −1, a lymphatic endothelial marker, of the ear sections from mice described in ( a ). Nuclei were counterstained with Hoechst 33342. Pearsons’s co-localization index (PC) was calculated using 4 images of three Prox1 flox/flox and four Prox1 flox/flox ; Cdh5-CreER T2 . PC = 0.088 in control mice (N3); PC = 0.640 in Prox1-depleted mice (N = 4); p = 0.01 (t-test). ( d,e ) LECs were transfected with the indicated siRNAs and after 72 h whole cell extracts were analysed by RTqPCR ( d ) for the indicated targets with GAPDH as an internal control. Bars represent an average of three independent experiments, error bars show SD across the experiments. ( e ) Immunoblotting for the indicated proteins using γ-tubulin (TUBG1) as a loading control; numbers indicate the intensity of MMP14 band for each sample normalized to the corresponding loading control. Cropped membranes are shown, uncropped membranes can be found in Fig. . ( f,g ) HEK293FT cells were transfected with the indicated siRNAs, 32 h later whole cell extracts were analysed by RTqPCR ( f ) for the indicated targets with ACT as an internal control. Bars indicate the average of two independent experiments, error bars show SD; and by immunoblotting ( g ) for the expression of the indicated proteins using γ-tubulin (TUBG1) as a loading control; numbers indicate the intensity of MMP14 band for each sample normalized to the corresponding loading control. Cropped membranes are shown, uncropped membranes can be found in Fig. . **p < 0.01; ***p > 0.001.

Article Snippet: For the staining of mouse ears the following antibodies were used: rat anti-mouse PECAM1 (BD-Biosciences; 553370), rabbit anti-mouse LYVE-1 (103-PA50AG, Reliatech), goat anti-PROX1 (R&D AF2727), rat anti mouse endomucin (Santa Cruz sc-65495), hamster anti mouse podoplanin (Developmental Studies hybridoma bank 8.1.1) and mouse monoclonal anti-MMP14 (LEM clone; Millipore).

Techniques: Expressing, In Vivo, In Vitro, Immunofluorescence, Immunohistochemistry, Marker, Transfection, Western Blot

Journal: Scientific Reports

Article Title: PROX1 is a transcriptional regulator of MMP14

doi: 10.1038/s41598-018-27739-w

Figure Lengend Snippet: PROX1 silencing increases MMP14 expression and MMP14-dependent 3D sprouting in cancer cells. ( a ) Chromatin immunoprecipitation in HepG2 and SW620 cells ectopically expressing Myg-tagged PROX1. Chromatin was immunoprecipitated using either an anti-Myc antibody or an irrelevant anti-HA antibody and subsequently amplified by qPCR for the indicated regions of the MMP14 promoter (illustrated in Fig. ). Average fold enrichment over the anti-HA control is shown for three independent experiments; error bars represent SD. ( b ) HepG2 and SW620 cells were transfected with the indicated siRNAs for 48 h and whole cell extracts were analysed by RTqPCR for the indicated targets with GAPDH as an internal control. Bars represent an average of two independent experiments, error bars show SD across the experiments. ( c ) HepG2 and SW620 cells were treated as in ( b ) and whole cell extract was analysed by immunoblot for the indicated proteins using actin as a loading control; numbers indicate the intensity of the MMP14 band normalized to the corresponding loading control. Cropped membranes are shown, uncropped membranes can be found in Fig. . ( d , e ) 3D fibrin invasion assay with HepG2 cells transfected twice in consecutive days with the indicated siRNAs and embedded in 3D fibrin in the presence of either DMSO or 50 μM of the MMP14 inhibitor NSC405020. After 4 days, fibrin gels were fixed and stained with Phalloidin (Phall A488) and Hoechst 33342. ( d ) Representative images are shown. ( e ) Quantification of three images per condition from two independent experiments (n > 100 cells/condition). Bars represent the average area occupied by each cell cluster and normalized to the scr siRNA and DMSO-treated sample. n.s.: non-significant; **p > 0.01; ***p > 0.001.

Article Snippet: For the staining of mouse ears the following antibodies were used: rat anti-mouse PECAM1 (BD-Biosciences; 553370), rabbit anti-mouse LYVE-1 (103-PA50AG, Reliatech), goat anti-PROX1 (R&D AF2727), rat anti mouse endomucin (Santa Cruz sc-65495), hamster anti mouse podoplanin (Developmental Studies hybridoma bank 8.1.1) and mouse monoclonal anti-MMP14 (LEM clone; Millipore).

Techniques: Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Transfection, Western Blot, Invasion Assay, Staining

Journal: Scientific Reports

Article Title: PROX1 is a transcriptional regulator of MMP14

doi: 10.1038/s41598-018-27739-w

Figure Lengend Snippet: PROX1 reintroduction decreases MMP14 expression and reduces 3D invasiveness in BEC and cancer cells. ( a ) MDA-MB-231 and HuAR2T were treated with the indicated siRNAs for 72 h, and whole cell extracts were analysed by immunoblotting for the indicated proteins using actin as a loading control; numbers indicate the intensity of MMP14 band for each sample normalized to the corresponding loading control. Cropped membranes are shown, uncropped membranes can be found in Fig. . ( b ) The samples in ( a ) were analysed by RTqPCR for the indicated targets with GAPDH as an internal control. Bars represent the average of three independent experiments, error bars show SD. ( c ) 3D fibrin invasion assay with MDA-MB-231 transduced with lentiviruses expressing PROX1 WT or MUT and stained with Phalloidin (Phall A488) and Hoechst. Empty vector was used as a control (mock). Representative images are shown. ( d ) Quantification of three images per condition described in ( c ) from three independent experiments (n > 200 cells/condition). Bars represent the average area occupied by each cell and normalized to the mock infected cells. (e) 3D sprouting assay of HuAR2T spheroids, treated and stained as in ( c ). Representative images are shown. (f) Quantification of three independent experiments described in ( e ). Bars represent the average of the total area of each spheroid divided by the area of the non-invading cells. Three spheroids per condition were quantified in three independent experiments. Error bars indicate SD. (g,h) MDA-MB-231 cells were transduced with PROX1 WT-expressing lentivirus for 24 h and subsequently transfected with a control vector (mock) or MMP14 expression vector for 24 h. Cells were then embedded in fibrin and treated as in ( c ). (g) Representative enlarged images are shown. (h) Quantification of three images/condition for two independent experiments (n > 100). Bars represent the average of the total area occupied by each cell and normalized to the mock transfected cells. Error bars indicate SD. **p < 0.005; ***p > 0.001.

Article Snippet: For the staining of mouse ears the following antibodies were used: rat anti-mouse PECAM1 (BD-Biosciences; 553370), rabbit anti-mouse LYVE-1 (103-PA50AG, Reliatech), goat anti-PROX1 (R&D AF2727), rat anti mouse endomucin (Santa Cruz sc-65495), hamster anti mouse podoplanin (Developmental Studies hybridoma bank 8.1.1) and mouse monoclonal anti-MMP14 (LEM clone; Millipore).

Techniques: Expressing, Western Blot, Invasion Assay, Transduction, Staining, Plasmid Preparation, Infection, Transfection