mmp14 inhibitor nsc405020  (Tocris)

Journal: Nature immunology

Article Title: Fungal sensing by dectin-1 directs the non-pathogenic polarization of T H 17 cells through balanced type I IFN responses in human DCs.

doi: 10.1038/s41590-022-01348-2

Figure Lengend Snippet: Fig. 7 | Dectin-1-induced release of active TGF-β requires MMP14 activity. a, SEAP assay on supernatant of HEK-Blue TGF-β reporter cells for quantification of active TGF-β in the supernatant of unstimulated DCs or after stimulation of DCs with curdlan or C. albicans, in the presence of MMP14 inhibitor NSC405020 at 24 h (n = 3). b–e, FRET assay of extracellular MMP14 activity (RFU) in unstimulated DCs or after stimulation of DCs with curdlan or C. albicans, in the presence of NSC405020 (b), blocking IFN-α/βR antibodies (c), a concentration

Article Snippet: DCs were preincubated for 2 h with MMP14 inhibitor NSC405020 (100 μM; Tocris) or blocking antibodies, anti-dectin-1 (20 μg ml−1; clone 259931, MAB1859, R&D Systems), anti-IFN-α/βR2 (20 μg ml−1; clone MMHAR-2, PBL Assay Science), anti-αvβ1 (10 μg ml−1; clone P5D2, MAB17781, R&D Systems), anti-αvβ3 (10 μg ml−1; clone 23C6, MAB3050, R&D Systems), anti-αvβ5 (10 μg ml−1; clone P5H9, MAB2528, R&D Systems), anti-αvβ6 (10 μg ml−1; clone 10D5, ab77906, Abcam), anti-αvβ8 (10 μg ml−1; kind gift from S.L.

Techniques: Activity Assay, SEAP Assay, Blocking Assay, Concentration Assay

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: The MMP14 inhibitor NSC405020 was obtained from Sigma-Aldrich (2044451).

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: The MMP14 inhibitor NSC405020 was obtained from Sigma-Aldrich (2044451).

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: The MMP14 inhibitor NSC405020 was obtained from Sigma-Aldrich (2044451).

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: The MMP14 inhibitor NSC405020 was obtained from Sigma-Aldrich (2044451).

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: The MMP14 inhibitor NSC405020 was obtained from Sigma-Aldrich (2044451).

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