mmp14 inhibitor nsc 405020  (MedChemExpress)

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Combined transcriptomic and proteomic analyses identified MMP14 as a key molecule in AMLMSC supporting leukemia cell growth. A , B . Analysis of apoptosis in primary leukemia cells after 48 h of co-culture with five cases each of AML-MSC and HD-MSC, using the Annexin V APC Apoptosis Detection Kit ( n = 5). Alone, leukemia cells cultured alone; Co, leukemia cells cocultured with MSCs. C , D . Flow BrdU assay for assessing the proliferation of primary leukemia cells after 48 h of co-culture with 5 AMLMSC and 5 HDMSC cases( n = 5). E . Volcano plots displaying the expression of differentially expressed genes between five AML-MSC and five HD-MSC samples, with purple representing upregulation and blue representing downregulation( n = 5). F . Enrichment analysis of differentially expressed genes. G . Proteomic analysis of supernatants from 4 AML-MSC and 4 HD-MSC samples, including a heat map of differentially expressed proteins( n = 4). H . Intersection analysis of differentially expressed genes identified in transcriptomic and proteomic analyses. I . Kaplan-Meier survival analysis of OHSU-AML dataset revealed that high expression of MMP14 is associated with poorer overall survival. J . PCR detection of MMP14 expression in AMLMSC ( n = 12) compared to HDMSC ( n = 6). K . Co-immunohistochemical staining of MMP14 (red) and CD271 (green) in AML and healthy control bone marrow samples. Scale bar = 20 μm ( n = 5)

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Co-Culture Assay, Cell Culture, BrdU Staining, Expressing, Immunohistochemical staining, Staining, Control

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Effects of MMP14 on MSC proliferation and cytokine secretion. A , B . Knockdown efficiency of MMP14 in MSCs verified by Western blot and RT-qPCR. C , D . Representative plots of CFU-F in the MMP14 knockdown and control groups. Quantification of CFU-F colonies in MSCs. E , F . BrdU assay showing cell proliferation rates in the MMP14 knockdown and control groups. G , H . Cell cycle analysis of the MMP14 knockdown and control groups. I - K . After co-culturing Kasumi-1 and Molm-13 cells with MSCs from the knockdown and control groups for 48 h, flow sorting was performed on the MSCs followed by PCR analysis of TGF-β1, CXCL12, CXCL10, OPN, and COX-2 gene expression levels

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Control, BrdU Staining, Cell Cycle Assay, Gene Expression

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Knockdown of MSC-derived MMP14 significantly inhibits AML progression in vitro. A - C . Flow cytometry analysis of apoptosis rates in Kasumi-1 and Molm-13 cells co-cultured with the knockdown and control groups for 48 h. D - F . Flow cytometry PI analysis of cell cycle changes in Kasumi-1 and Molm-13 cells co-cultured with the knockdown and control groups for 48 h. G - I . Flow cytometry BrdU analysis of cell proliferation rates in Kasumi-1 and Molm-13 cells co-cultured with the knockdown and control groups for 48 h

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Knockdown, Derivative Assay, In Vitro, Flow Cytometry, Cell Culture, Control

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Effect of MMP14 deletion in MSCs on disease progression and leukemic stem cells in MLL-AF9 leukemic mice. ( A ) The experimental layout involved transplanting MLL-AF9 cells via the tail vein and MSCs from MMP14 knockdown and control groups via intra-femoral injection into mice. AML progression was monitored by flow cytometric analysis. ( B ) Survival rates of mice in MMP14 knockdown and control groups after cell transplantation ( n = 7). ( C ) Live imaging was performed on days 14, 21, and 28 post-transplant to monitor disease progression and burden( n = 5). D , E . Flow cytometry plots of BM LSCs and statistical analysis of the proportion of LSC in whole BM ( n = 4). F . Statistical analysis of the proportion of leukemia cells in mice of both groups ( n = 4). G . Bone marrow cell count statistics in both groups of mice ( n = 4). H , I . Representative flow cytometry plots of LSC apoptosis and statistical analysis of the apoptosis rate of LSCs ( n = 4). J , K . Representative flow cytometry plots of the LSC cell cycle and statistical analysis of the percentage of apoptosis in LSCs ( n = 4)

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Knockdown, Control, Injection, Transplantation Assay, Imaging, Flow Cytometry, Cell Counting

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: MMP14 exerts its functions by activating the JAK-STAT pathway in AML cells through the secretion of PGE2. ( A ) Transcriptome analysis of Kasumi-1 cells co-cultured with MSCs from the MMP14 knockdown and control groups after 48 h. Volcano plots are shown. ( B ) Heat map of differentially expressed genes. ( C ) Enrichment analysis of differentially expressed genes. ( D ) GSEA analysis showing that MMP14 knockdown in MSCs is associated with inhibition of the JAK-STAT pathway. ( E ) Western blot analysis of STAT3, P-STAT3, STAT5, and P-STAT5 protein levels in Kasumi-1 and Molm-13 cells co-cultured with MSCs from the MMP14 knockdown and control groups. ( F ) ELISA assay of PGE2 levels in the supernatant of MSCs from the MMP14 knockdown and control groups. ( G ) ELISA assay of PGE2 levels in the serum of mice from the MMP14 knockdown group and the control group. H-J. Proliferation of Kasumi-1 and Molm-13 cells in the presence or absence of PGE2 (1 μm) in the co-culture system of the knockdown and control groups

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Cell Culture, Knockdown, Control, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: MMP14 confers chemoresistance to Ara-C. A . Overview of the experimental design, dividing mice into four groups based on the presence or absence of the inhibitor (NSC-405020) and cytarabine. B . Survival statistics of mice ( n = 7). C - E . Representative FACS analysis and statistical proportions of leukemia cells in the four groups of mice ( n = 4). F . Representative images of Wright-Giemsa staining of bone marrow cells and hematoxylin-eosin staining of spleens. G . Representative images of mouse spleens. H - J . Immunohistochemical staining of P-STAT3 and P-STAT5 in mouse bone marrow and quantification of the staining. Scale bar, 50 μm

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Staining, Immunohistochemical staining

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Inhibition of MMP14 suppressed AML patient blasts. A - C . Flow cytometry analysis of apoptosis rates in primary leukemia cells co-cultured with the knockdown and control groups for 48 h. Alone, leukemia cells cultured alone; Co, leukemia cells cocultured with MSCs. D , E . Viable cells were counted using trypan blue staining and assessed with a hemocytometer after 48 h of co-culturing. F . Schematic of the PDX model construction process. Primary human leukemia cells were isolated from high-leukemia AML patients using leukapheresis and PBMC separation. These cells were then injected into NSG mice via the tail vein. Four weeks later, one mouse per week was randomly sacrificed, and the proportion of leukemia cells in the BM was analyzed by flow cytometry. Drug treatment commenced when the leukemia cell proportion reached ≥ 20%. Following successful PDX model construction, mice were randomly allocated to four treatment groups: DMSO-treated control (Ctrl), MMP14 inhibitor (inhibitor), cytarabine (Ara-C), and a combination of MMP14 inhibitor and cytarabine (inhibitor + Ara-C). G . Survival statistics for the mice were compiled ( n = 6). H-J. Flow cytometric representative plots and statistical analysis detailing the proportion and absolute numbers of human CD45 + cells in the four mouse groups ( n = 5)

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Inhibition, Flow Cytometry, Cell Culture, Knockdown, Control, Staining, Isolation, Injection

Journal: Experimental Hematology & Oncology

Article Title: MMP14 from BM-MSCs facilitates progression and Ara-C resistance in acute myeloid leukemia via the JAK/STAT pathway

doi: 10.1186/s40164-025-00635-6

Figure Lengend Snippet: Mechanism of action of MSC-derived MMP14 in promoting AML progression. The model illustrates how MSC-derived MMP14 promotes AML cell growth and chemotherapy resistance through the secretion of PGE2, activating the JAK-STAT pathway, leading to AML progression. Additionally, MSC-derived MMP14 enhances MSC proliferation and self-renewal, while also inducing cytokine alterations

Article Snippet: The MMP14 inhibitor NSC 405020 was purchased from MCE (HY-15827, China).

Techniques: Derivative Assay

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: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: Expression and localization of MMP14 prior to and during EMT of chick NC cells.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: Expressing

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: A, diagram showing the main steps of the Cornish pasty culture technique. B-C, immunostaining for Snail2 on cryosections of embryos treated with DMSO (B) or MMP14 inhibitor NSC405020 at 0.5mM (C). D, plot showing the size of the Snail2-positive region normalized to DMSO in the dorsal neural tube (DMSO: n embryos =4, n sections =8; NSC: n embryos =7, n sections =7, from one experiment). Error bars show the standard deviation. E, distance of migration from the dorsal midline (DMSO: n embryos =11, n sections =11; NSC: n embryos =11, n sections =11, from two experiments). Error bars represent the standard deviation. ANOVA, followed by multiple comparisons; **, p value <0.01. F, in situ hybridization using a mix of probes against Foxd3 and Sox10 to stain the cephalic NC cells after electroporation of control scrambled siRNA (first column, n=28 embryos from 7 experiments), siRNA-MMP14 alone (second column, n=46 embryos from 11 experiments), siRNA-MMP14 and full length MMP14 (third column, n=17 embryos from 6 experiments), siRNA-MMP14 and the delta-catalytic form (fourth column, n=11 embryos from 3 experiments), siRNA-MMP14 and the inactive point mutant (fifth column, n=14 embryos from 2 experiments), siRNA-MMP14 and the delta-cytoplasmic form (sixth column, n=11 embryos from 3 experiments). Dotted lines indicate the level of the sections shown below each whole mount. G, plot of the area occupied by Foxd3/Sox10-positive NC cells above the post-otic neural tube from whole mount images. ANOVA with Kruskal-Wallis multiple comparisons, ***p<0.0005. H, immunostaining against Snail2 after electroporation of scrambled siRNA, siRNA-MMP14 and co-electroporation with siRNA-MMP14 and full length MMP14. Scrambled and siRNA are in green (GFP), MMP14-FL-mCherry is in magenta. I, plot of the NC area (Snail2) with scrambled (n embryos =11, n sections =132), siRNA-MMP14 (n embryos =24, n sections =303) and siRNA-MMP14 and full length MMP14 (n embryos =3, n sections =15), ANOVA with Kruskal-Wallis multiple comparisons, ****p<0.0001. J-K, neural tube explants cultured on fibronectin coated dishes counterstained with SiR-DNA. Cells electroporated with scrambled siRNA or SiRNA-MMP14 are GFP-positive. L, ratio of GFP-positive cells over the total number of cells counted from SiR-DNA staining, n scrambled =7 explants, n siRNA MMP14 =9 explants from 4 independent experiments. Unpaired t-test with Welsh’s correction, p=0.1186.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: Immunostaining, Standard Deviation, Migration, In Situ Hybridization, Staining, Electroporation, Mutagenesis, Cell Culture

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: A-B, in situ hybridization for chick MMP14 after Ets1 overexpression (A) or Snail2 overexpression (B) in cephalic regions, n=5 embryos each. Arrowhead indicates ectopic activation of MMP14. C, Immunostaining for MMP14 (magenta) after Ets1 overexpression (GFP) in the trunk. Nuclei are stained with DAPI. Arrows indicate the loss of apical MMP14, arrowheads indicate regions of basolateral accumulation of MMP14, n=3 embryos.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: In Situ Hybridization, Over Expression, Activation Assay, Immunostaining, Staining

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: A-D, in situ hybridization and transversal cryosections through the post-otic NC region for Cadherin-6B (A), Cadherin-7 (B), RhoB (C) and Integrin-β3 (D) in embryos electroporated with scrambled (left whole mount, top section) or MMP14 (right whole mount, bottom section) siRNA, n scrambled =23, n siRNA-MMP14 =22 ; from 2 experiments. Asterisks on sections mark the electroporated side, insets in whole mount images indicate the electroporated regions. Dotted line delineate the midline of the neural tube. Arrowheads indicate NC cells located within the dorsal neural tube expressing the gene of interest.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: In Situ Hybridization, Expressing

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: A-B, immunostaining against E-cadherin (red) and Snail2 (white) in embryos electroporated with scrambled (A, n=5 embryos) or MMP14 (B, n=3 embryos) siRNA. C-D, immunostaining against N-cadherin (red) and Snail2 (white) in embryos electroporated with scrambled (C, n=5 embryos) or MMP14 (D, n=3 embryos) siRNA. E-F’, immunostaining against pericentriolar material 1 (PCM1) and AP2 (white) in embryos electroporated with scrambled (E, n=6 embryos) or MMP14 (F-F’, n=5 embryos) siRNA. Arrowheads in E indicate basolateral localization of PCM1 on both the non-electroporated and the scrambled-siRNA sides. Arrowheads in F marks the basolateral localization of PCM1 on the non-electroporated side only. Note that PCM1 is solely found within the apical domain in AP2-positive cells after electroporation of siRNA-MMP14 (bracket). F’, zoom on the neural crest domain. Electroporated cells are visualized by GFP expression (green). Data from 2 independent experiments.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: Immunostaining, Electroporation, Expressing

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: Basolateral localization of MMP14 is sufficient to impair apicobasal polarity independently of its catalytic activity

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

doi: 10.1101/402180

Figure Lengend Snippet: Basolateral localization of MMP14 contributes to EMT by destabilizing apicobasal polarity.

Article Snippet: MMP14 inhibitor NSC 405020 (EMD Millipore, 444295).

Techniques:

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) mRNA expression of a panel of LEC markers ( CD34 , PROX1 , FLT4 ) in 3D LEC primed Bowes and WM852 cells (*) before the cells were used for the in vivo xenograft assay. Expression in monotypic-cultured LEC was used as a control and set to one. ( b ) End point analysis of the volume (left panel) and weight (right panel) of the Bowes/Bowes* and WM852/WM852* tumors. A dot represents one mouse. Error bars indicate s.e.m. *: p<0.05. n.s., non-significant ( c ) Immunohistochemistry of xenograft sections from the LEC co-cultured WM852* (left panels) or Bowes* (right panels) stained for mouse Lyve-1 (red) to detect mouse lymphatic vessels and human MMP14 (green) as a marker of the human melanoma cells. Nuclei were counterstained with Hoechst 33342. Scale bar = 100 µm. ( d, e ) Luciferase signal from lymph nodes isolated from mice bearing WM852 (n = 7) and WM852*(n = 8) ( d ), or Bowes and Bowes* (n = 8) ( e ); each line shows lymph nodes from one mouse derived tumors. ( f ) Quantitative-PCR (q-PCR) for human Alu sequences from lung genomic DNA isolated from mice bearing WM852 and WM852* derived tumors. Each dot in the graph represents the luciferase signal intensity obtained from one isolated organ per mouse, and the red line marks the median of the samples. ( g ) Luciferase signal from liver and lungs isolated from mice bearing Bowes or Bowes* derived tumors (n = 8); each box represents an organ from one mouse.

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Expressing, In Vivo, Xenograft Assay, Cell Culture, Control, Immunohistochemistry, Staining, Marker, Luciferase, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Right panels: representative confocal images of MMP14 (red) expression in WM852 and Bowes co-cultured with LECs (*, upper panels) and from monotypic culture (bottom panels). Nuclei were counterstained with Hoechst 33342. GFP-expressing melanoma cells are shown white in the inset. Arrowheads indicate MMP14 localization to the cell-cell contacts. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. Left panel: quantification of MMP14 intensity analysed in four images per condition from two independent experiments. More than 100 cells were always analysed per condition. Average is shown, error bars represent SD; *: p<0.05. n.s., non-significant. ( b ) Quantification of the 3D sprouting index of WM852 and WM852* treated with the indicated siRNAs for 72 hr followed by magnetic separation and the 96 hr fibrin assay. The graph represents the average of three images per condition in each of the two independent experiments, error bars indicate SEM; *: p<0.05. n.s., non-significant. ( c ) Representative confocal images of MMP14 (green) and Notch3 (red) in WM852 and WM852*. Arrowheads indicate the cell-cell junction where MMP14 and Notch3 co-localize. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC–melanoma (below the line; GFP positive cells (white) in the inset) border. Scale bar = 50 µm. ( d ) mRNA fold change of the indicated targets in WM852 and WM852* upon treatment with the indicated siRNA for 72 hr and following magnetic separation. Graphs show the average of three independent experiments, error bars indicate SEM, *: p<0.05; **: p<0.01. n.s., non-significant ( e ) Representative confocal images of Notch3 staining (red) in WM852* treated with the indicated siRNAs for 72 hr. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the LEC-WM852 (GFP positive cells (white) in the inset) border. Scale bar = 50 µm. ( f ) Quantification of Notch3 signal intensity of WM852* treated as in ( e ) and described in ( a ). Error bars indicate SD; *: p<0.05. Full size confocal images are available as .

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Expressing, Cell Culture, Staining

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Flow cytometry analysis of MMP14 levels in WM852 and WM852*. Mean fluorescence intensity normalized to WM852 is shown, error bars represent SD across three independent experiments; *: p<0.05. ( b ) Left panels: representative images of MMP14 staining (red) in WM165 and WM793 cells (GFP positive cells (white) in the insets) cultured in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. Right panel: quantification of MMP14 signal intensity for the indicated cell lines. Average of the intensity is shown from four images/condition from two independent experiments. More than 100 cells were always analysed per condition. Error bars indicate SD. n.s., non-significant ( c ) Confocal images of Bowes (upper panels) and WM852 (lower panels) stained with TGN46 (green) and MMP14 (red) antibodies, nuclei were counterstained with Hoechst 33342. Scale bar: 25 µm. ( d ) Relative sprouting index of WM852 and WM852* treated with the vehicle (DMSO) or panMMP inhibitor GM6001 for 48 hr during co-culture and after magnetic separation as well as during the 3D fibrin assay. Error bars indicate SEM; **: p<0.01. ( e ) MMP14 mRNA fold change in WM852 and WM852* treated with the indicated siRNAs for 72 hr; *: p<0.05; **: p<0.01. n.s., non-significant. Full size confocal images are available as .

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Flow Cytometry, Fluorescence, Staining, Cell Culture, Co-Culture Assay

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Representative enlargement with separated channels of the images in . Merge, Notch3 (red) and MMP14 (green) stainings are shown. ( b ) MMP14 mRNA fold change in WM852* treated with the indicated siRNAs for 72 hr. Data averaged from two independent experiments is shown. n.s., non-significant. ( c ) Right panels: representative confocal images of MMP14 (red) in WM852* after the cells were treated with indicated siRNAs for 72 hr. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate LEC-melanoma (GFP expressing cells (white in the inset) border. Scale bar = 50 µm. Left panel: quantification of the MMP14 intensity analysed in four images per condition from two independent experiments. More than 100 cells were always analysed per condition. Average is shown, error bars represent SD. n.s., non-significant ( d ) Immunoblot using anti-Notch3 (upper panels) and anti-actin (lower panels) antibodies of WM852 and WM852* (+LEC) treated as indicated and subjected to magnetic separation prior to sample preparation. Notch3 FL: full length Notch3; NICD3: Notch3 intracellular domain. Panels represent the bands from the same membrane and same exposure time. ( e ) Relative Notch3 and cleaved NICD3 band intensity normalized to actin of two independent experiments performed as in ( d ). Average is shown, error bars represent SD; *: p<0.05. Full size confocal images are available as .

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Expressing, Western Blot, Sample Prep, Membrane

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Representative confocal images of active β1-integrin (12G10) staining (red) in the indicated melanoma cell lines (GFP positive cells (white) in the inset) in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma border. Scale bar = 50 µm. ( b ) Quantification of 3D sprouting index in WM852 and WM852* mock treated or treated with β1-integrin blocking antibody (AIIB2) during the 96 hr fibrin growth assay. Graph shows the average of at least three images per condition per two independent experiments, error bars indicate the SEM; *: p<0.05. n.s., non-significant. ( c ) Representative confocal image of active β1-integrin (12G10, red) and MMP14 (green) staining of WM852* (white cells in the inset). Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the border between LEC and WM852 (white, GFP positive WM852 cells in the inset). The right and bottom panels show an enlargement of the area enclosed within the white square as a merge, Notch3 (red) and MMP14 (green) in separated channels. Scale bar = 50 µm. ( d,f ) Representative confocal images of WM852* treated with the indicated siRNAs for 72 hr and stained for active β1-integrin with 12G10 (d, red), or total β1-integrin with P5D2 (f, red) antibodies. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the LEC-WM852 borders (white, GFP positive WM852 cells in the inset) border. Scale bar = 50 µm. Quantification of the average 12G10 ( e ) and total β1-integrin ( g ) signal intensity in WM852* (white) cells. Four images/condition were quantified from two independent experiments. More than 100 cells were always analysed per condition; error bars indicate SD. *: p<0.05. n.s., non-significant. Full size confocal images are available as a .

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Staining, Blocking Assay, Growth Assay

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a, b ) Left and middle panels: representative confocal images of active β1-integrin expression using 12G10 ( a ) and 9EG7 antibodies (red) in WM852* cells treated with indicated siRNAs for 72 hr. Nuclei were counterstained with Hoechst 33342. GFP-expressing melanoma cells are shown white in the inset. The dashed line indicates the LEC-melanoma border. Scale bar = 50 µm. Right panels: Quantification of the average of 12G10 ( a ) and 9EG7 ( b ) signal intensities. Four images/condition were quantified from two independent experiments. More than 100 cells were always analysed per condition; error bars indicate SD. n.s., non-significant. ( c ) mRNA fold change of the indicated targets in WM852 and WM852* following treatment with AIIB2 β1-integrin blocking antibody for 48 hr and magnetic separation. Graphs show the average of two independent experiments, error bars indicate SD; *: p<0.05; n.s., non-significant. ( d,e ) Left and middle panels: Representative confocal images of control and AIIB2 β1-integrin blocking antibody treated WM852* cells stained with Notch3 (d, red), or MMP14 (e, red) antibodies. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the LEC-WM852 border (white, GFP positive WM852 cells in the inset) border. Scale bar = 50 µm. Right panels: Quantification of the average Notch3 ( d ) and MMP14 ( e ) signal intensity in WM852* (white) cells as described in ( a,b ); error bars indicate SD. n.s., non-significant. Full size confocal images are available as a .

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Expressing, Blocking Assay, Control, Staining

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet:

Article Snippet: Gamma-secretase inhibitor DAPT (Sigma) and pan MMP inhibitor GM6001 (Tocris Biosciences) at 10 μM concentrations, MMP14 hemopexin domain inhibitor NSC 405020 (Selleckchem) at 50 μM and the β1-integrin blocking antibody AIIB2 were applied to the growth medium during the 48 hr LEC-WM852 2D co-cultures and also to the 96 hr 3D fibrin assays when indicated.

Techniques: Functional Assay, Transfection, Construct, Plasmid Preparation, Sequencing, SYBR Green Assay, Software