Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells). SMCs alone, with direct contact and without direct contact to isolated myenteric plexus cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Isolation

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Immunofluorescence staining of enteric nervous system (ENS) cells co-cultured with smooth muscle cells in a three-dimensional HyStem-C hydrogel, showing ( a ) glial fibrillary acidic protein (GFAP)–positive glial fibers (magenta), ( b ) smooth muscle cells stained for smooth muscle actin (SMA, cyan), ( c ) neuronal fibers labeled with βIII-tubulin (Tuj1, green), and ( d ) merged image. Cell nuclei are counterstained with DAPI (blue). Non-specific staining observed in the SMA channel indicates the presence of additional cell types, likely fibroblasts. Scale bar 50 μm. ( e ) higher-magnification image of a GFAP-positive glial cell (magenta). Scale bar 100 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Immunofluorescence, Staining, Cell Culture, Labeling

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Contracting muscle fibers in the live cultures of muscle cells together with ENS cells in HyStem-C Hydrogel (real time): ( a ) thin muscle fibers, ( b ) thick muscle fibers. Light microscopy.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Light Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Smooth muscle and ENS cells in 3D scaffolds (14 days) co-cultured in 3D scaffold, showing neurons in green (ß-Tubulin III), muscle cells in red (smooth muscle actin (SMA)) and nuclei in blue (DRAQ5) ( a ) Confocal microscopy lower Magnification, ( b ) Confocal microscopy 3D structure in merged channels view from the side in higher magnification (Scale bars 50 μm) and ( c ) Confocal microscopy, separated channels view from above in higher magnification, ( d ) Electron microscopy of two muscle cells in close contact within the thickness of the three-dimensional matrix. The cells have large nuclei, an elongated shape, and actin microfilaments typical for muscle cells. Scale bar 2 μm, ( e ) Electron microscopy of plasma membrane of a muscle cell, with arrows indicating caveolae. Scale bar 200 nm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Cell Culture, Confocal Microscopy, Electron Microscopy, Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Images of 3 weeks old co-culture of ENS cells and muscle cells in different layouts: ( a ) SMCs alone, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells Scale bars 100 μm, ( d ) paracrine interaction between SMCs and ENS in higher magnification, ( e ) Direct contact between smooth muscle and ENS cells in higher magnification.– muscle cells Scale bars 20 μm, ( f ) 3D reconstruction of muscle fibers in confocal microscopy by the direct contact between SMCs and ENS, ( j ) neurons, which are intercommunicated in the neuronal net within the muscle layer by the direct contact between SMCs and ENS. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells. Scale bars 50 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Imaging of the whole thickness of the 3D scaffolds after 3 weeks of ENS cells and muscle cells co-culture in different layouts: ( a ) SMCs without ENS cells, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Imaging, Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Confocal microscopy images of smooth muscle and ENS cells co-cultured in 3D scaffolds (14 days of culture). Secretory vesicles and synapses are labelled in green (Synaptobrevin 2), while muscle cells in red (smooth muscle actin (SMA)). Scale bars 20 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Confocal Microscopy, Cell Culture

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells). SMCs alone, with direct contact and without direct contact to isolated myenteric plexus cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Isolation

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Images of 3 weeks old co-culture of ENS cells and muscle cells in different layouts: ( a ) SMCs alone, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells Scale bars 100 μm, ( d ) paracrine interaction between SMCs and ENS in higher magnification, ( e ) Direct contact between smooth muscle and ENS cells in higher magnification.– muscle cells Scale bars 20 μm, ( f ) 3D reconstruction of muscle fibers in confocal microscopy by the direct contact between SMCs and ENS, ( j ) neurons, which are intercommunicated in the neuronal net within the muscle layer by the direct contact between SMCs and ENS. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells. Scale bars 50 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Imaging of the whole thickness of the 3D scaffolds after 3 weeks of ENS cells and muscle cells co-culture in different layouts: ( a ) SMCs without ENS cells, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Imaging, Co-Culture Assay, Confocal Microscopy

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay