Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: B16F1 cell proliferation and migration on MMP14 Sf−/− and MMP14 Sf+/+ fibroblast matrix. ( a ) Schematic representation of fibroblast culture to obtain their matrix (picture of Coomassie-stained matrix after fibroblast removal) and seeding B16F1 cells on it. ( b ) BrdU incorporation measurement of B16F1 cells cultured on fibroblast matrix. ( c ) Colony outgrowth assay showing melanoma migration on fibroblast matrix (left) and quantification (right). Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix) n = 3; mSF −/− (MMP14 Sf−/− matrix) n = 3; continuous line, cell front after 0 h; dashed line, cell front after indicated period of time; * p < 0.05; *** p < 0.001; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Migration, Staining, BrdU Incorporation Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of MMP14 Sf−/− and MMP14 Sf+/+ fibroblast matrices. ( a ) Mass Spectrometric and ( b ) immunoblot analysis of fibroblast matrix and conditioned medium (8% SDS-PAGE). ( c ) Transcriptional analysis of fibroblast lysates for collagens VI (COL6A1) and XIV (COL14A1). ( d ) In vitro processing of recombinant collagen XIV (Coll XIV) and human MMP14 visualized on immunoblot and silver staining (4–12% Bis-Tris gradient gel). Collagen XIV fragments generated by MMP14 are indicated with black arrows. MMP14 Sf+/+ (WT), n = 3; MMP14 Sf−/− (SF −/− ), n = 3; pro-MMP14, inactive form of MMP14; * p < 0.05.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Western Blot, SDS Page, In Vitro, Recombinant, Silver Staining, Generated

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Melanoma cells proliferation on collagen XIV coatings. BrdU incorporation analysis of B16F1 cells cultured on ( a ) collagen XIV (Coll XIV) (1 to 10 µg/mL) and ( b ) on serum (FCS) alone and in combination with collagen XIV (1 to 10 µg/mL). ( c ) Proliferation was measured in cells cultured on matrix from control fibroblasts (mWT) alone or with the addition of collagen XIV (1 to 10 µg/mL) and MMP14 Sf−/− (SF −/− ) fibroblasts. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix), n = 3; mSF −/− (MMP14 Sf−/− matrix), n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: BrdU Incorporation Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: B16F1 cell migration on MMP14 Sf+/+ fibroblast matrix enriched with collagen XIV. Colony outgrowth assay on MMP14 Sf+/+ fibroblast matrix (mWT) containing 10 µg/mL collagen XIV (Coll XIV). The FCS-coated surface was used as a control. Below is the quantification in the percentage of the migrated area. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix), n = 3; FCS, n = 3; a continuous line marks the cell border at time 0 h (this time point is shown in the insert); dashed line, cells border after indicated period of time; * p < 0.05; scale: 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of B16F1 cell adhesion on collagen XIV coating and fibroblast matrix. ( a ) Melanoma adhesion assay on collagen XIV (Coll XIV) coatings (1 µg/mL; 10 µg/mL), ( b ) wildtype fibroblast matrix (mWT), in combination with 10 µg/mL collagen XIV (mWT + Coll XIV) and MMP14 Sf−/− matrix (mSF −/− ). Fibronectin (FN) was used as positive control. Experiments were repeated three times. mWT (MMP14 Sf+/+ matrix, control matrix), n = 3, mWT + Coll XIV, n = 3; mSF −/− (MMP14 Sf−/− matrix), n = 3; * p < 0.05; **** p < 0.0001; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Cell Adhesion Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Collagen XIV expression in mouse melanoma. Immunofluorescence staining of collagen XIV (red) and DAPI (blue) in melanoma from MMP14 Sf+/+ and MMP14 Sf−/− mice. The graph represents quantified signal intensity per area measured within peritumoral tissue (100 µm radius from the tumor). MMP14 Sf+/+ (WT), n = 6; MMP14 Sf−/− (SF −/− ), n = 5; t, tumor; e, epidermis. ** p < 0.01; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Expressing, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth

doi: 10.3390/ijms222212276

Figure Lengend Snippet: Analysis of collagen XIV and MMP14 expression in human tissue samples. Immunofluorescence detection of collagen XIV (red) and immunohistochemical staining for MMP14 in human nevi and melanoma. The nuclei are in blue (DAPI). Average collagen XIV signal intensity per area is shown on the left. Yellow arrowheads, nests of melanocytic nevi; white/black arrowheads, cutaneous melanoma nests; e, epidermis; n, nevus; t, tumor; s, stroma; scale, 100 µm.

Article Snippet: Recombinant human MMP14 100 ng/µL (918-MPN, R&D Systems, Wiesbaden, Germany) was activated using 0.1 µg/mL trypsin (15090046, Thermo Fisher Scientific, Darmstadt, Germany) in 50 mM Tris, 0.15 M NaCl, 10 mM CaCl 2 , 5 µM ZnCl 2 , 0.05% Brij-35, pH 7.5 for 1 h at 37 °C.

Techniques: Expressing, Immunofluorescence, Immunohistochemical staining, Staining