Journal: Nature Communications

Article Title: T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration

doi: 10.1038/s41467-020-18586-3

Figure Lengend Snippet: a 3D-SIM of HUVEC lamellipodia, F-actin (yellow) and paxillin (magenta). Mature and nascent focal adhesions highlighted with cyan arrows and arrowheads, respectively, bar 5 µm. b Model of protrusion; nascent adhesions stabilize F-actin at the leading edge against opposing forces from membrane tension during a protrusion (orange membrane). c Example of ladder patterns of fibronectin (white) with gaps ranging from 4 to 16 µm coated with non-adhesive PLL-PEG (black). d Left, schematic: cells added to ladder patterns and protrude across gaps between fibronectin stripes as they spread. Right, HUVEC expressing Ftractin-mCitrine (yellow) added to fibronectin patterns (magenta). Bar, 50 µm. e Examples of cellular protrusions across different gap sizes. Smaller gaps of 4 µm, cells protrude broad lamellipodia. Larger gaps of 8 µm, cells utilize filopodia to initially protrude and then expand after making contact with the next fibronectin rung. Very large gaps of 12 µm cells, bleb and struggle to cross gaps or reach neighboring fibronectin rungs. f Images showing cellular behaviors depicted in ( e ). Lamellipodia, filopodia, and blebs are highlighted with cyan arrows, arrowheads, and asterisks, respectively, bar 10 µm. g The change in cell surface area one hour after first making contact with a stripe of fibronectin was quantified. Black bars represent mean and 95% confidence intervals ( n of 13, 23, 21, 31, 28, 26 cells for gaps of 4, 6, 8, 10, 12, and 16 µm, respectively from greater than three independent replicates), each gap size was compared to 4 µm using a one-way ANOVA with Dunnett’s multiple comparison test **** P < 0.0001. h Cells on patterns were fixed and stained for F-actin (yellow), paxillin (cyan); bar 5 µm. i Cells expressing Raichu Cdc42 FRET sensor (parula colormap, yellow and blue are 99th (high), 3rd (low) percentiles) were imaged on fibronectin patterns (white), bar 10 µm. j Model of protrusive actin-bundling playing an important role to compensate for lack of adhesion when cells protrude across non-adhesive gaps in order to reach the next available region of ECM.

Article Snippet: Pre-labeled rhodamine fibronectin was from Cytoskeleton Inc (FNR01).

Techniques: Membrane, Adhesive, Expressing, Comparison, Staining

Journal: Nature Communications

Article Title: T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration

doi: 10.1038/s41467-020-18586-3

Figure Lengend Snippet: a HUVEC expressing T-Plastin-mRuby3 and F-tractin-mCitrine on ladder patterns with 4 µm gaps, bar 20 µm. T-Plastin enrichment over F-tractin is shown as a parula colormap (yellow and blue are 99th (high), 3rd (low) percentiles). b Wildtype (WT) and T-Plastin KO1 HUVEC on fibronectin micropatterns were stained for F-actin (yellow) and paxillin (cyan), bars 10 µm. c 3 HUVEC T-Plastin KO lines (KO1-3) (red) change in area 1 h after first contact on a stripe with different sized gaps compared to WT (blue). ( n cells for Solid are: WT = 59, KO1 = 62, KO2 = 45, KO3 = 30; 4 µm gaps: WT = 20, KO1 = 29, KO2 = 61, KO3 = 74; 8 µm gaps: WT = 18, KO1 = 26, KO2 = 13, KO3 = 45; taken from ≥3 independent replicates). d Relative cell area changes over a 1 h period in both WT (blue) and all KO lines (red) used in ( c ). Means are shown as solid lines, transparent regions indicate the 95% confidence intervals. e WT and T-Plastin KO1 HUVEC were treated with MbCD ( n cells for WT and KO are: control 4 µm gaps 13 and 29, MbCD 4 µm 68 and 82, control 8 µm 21 and 31, MbCD 8 µm 15 and 18, respectively, controls are same from ( g ); taken from ≥3 independent replicates). f WT and KO1 HUVEC were pre-incubated on 8 µm gap patterns for 2 h prior to the indicated treatment. The change in the number of fibronectin stripes contacted 45 min after treatment is shown (n cells for WT and KO1 are: control 30 and 40, CK666 38 and 24, Bleb/Y27 38 and 36, ddH 2 O 30 and 28, respectively; taken from ≥3 independent replicates). Each condition was compared to WT control. g Similar to ( e ), but treated with cRGD ( n cells for WT and KO are: control 4 µm gaps 13 and 29, cRGD 4 µm 8 and 20, control 8 µm 21 and 31, cRGD 8 µm 15 and 15, respectively, controls are same from ( e ); taken from ≥3 independent replicates). h 3D invasion diagram. HUVEC are plated on top of collagen gel and invade. i WT and KO1 HUVEC stained for F-actin. Invading cells are marked by a cyan asterisk. Bars, 10 µm. j Quantification of collagen invasion. Black bars represent mean and SD. ( n slide wells for WT = 14 and KO1 = 15; taken from three independent replicates). Unless indicated elsewhere, black bars represent the mean and 95% confidence intervals, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant, analyzed using a one-way ANOVA with Sidak’s or Dunnett’s multiple comparison test for ( c , e , f , g ) or with an two-tailed unpaired t -test for ( j ).

Article Snippet: Pre-labeled rhodamine fibronectin was from Cytoskeleton Inc (FNR01).

Techniques: Expressing, Staining, Control, Incubation, Comparison, Two Tailed Test

Journal: Biochemical and biophysical research communications

Article Title: Overexpression of Snail in retinal pigment epithelial triggered epithelial-mesenchymal transition.

doi: 10.1016/j.bbrc.2014.02.119

Figure Lengend Snippet: Fig. 3. Overexpression of Snail in ARPE-19 cells induced EMT. ARPE-19 cells were transfected with pReceiver-Snail or pReceiver-control for 48 h. QRT-PCR and Immunoblotting were used to examine the expression of Snail, E-cadherin, ZO-1, a-SMA and fibronectin. (A) QRT-PCR analysis showed the increased Snail, fibronectin and a- SMA mRNA expression and decreased E-cadherin and ZO-1 mRNA expression. ⁄⁄P < 0.01 vs pReceiver-control. (B) Immunoblotting confirmed the expression of these EMT markers at protein levels.

Article Snippet: The primary antibodies used were as follows: 1:500 E-cadherin antibody and 1:1000 fibronectin antibody (R&D systems, Inc., USA), 1:1000 Snail antibody (Abcam Ltd., Cambridge, USA), 1:1000 a-SMA antibody (Sigma–Aldrich, MO, USA), 1:1000 ZO-1 antibody (Invitrogen, Carlsbad, CA), and 1:5000 GAPDH (Good HERE, Hangzhou, China).

Techniques: Over Expression, Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ATDC5 cells adhere more extensively to fibronectin, collagen I, and collagen IV. ATDC5 cells were screened with extracellular matrix array printed with collagen I (COL I), collagen III (COL III), collagen IV (COL IV), collagen V (COL V), collagen VI (COL VI), fibronectin (FN), vitronectin (VTN), laminin (LMN), tropoelastin (TE), and BSA as a negative control. (A) Representative bright-field images of ATDC5 cells incubated for 30 h indicated differential binding of a number of extracellular proteins. Scale bar: 40 μm. (B) Attached cell counts determined for each of the nine replicates, as well as mean and standard deviation are shown ( n = 9).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Negative Control, Incubation, Binding Assay, Standard Deviation

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Fibronectin interaction with graphene is stabilized by arginine residues. (A) Graphical rendering of the stabilized fibronectin atop the three graphene sheets with the four best arginine binders highlighted (Arg1166, Arg1369, Arg1374, Arg1403). The time evolution of the binding energy of these arginine residues with graphene is shown in the lower panel, color-coded for the amino acid residues. (B) Analogous to A but showing the data for the second studied configuration. This configuration features five arginine residue binders (Arg1166, Arg1351, Arg1379, Arg1445, Arg1493). (C) Binding energy with graphene computed for every amino acid with average binding energy above 1 kcal/mol, averaged over the 400 ns simulation. (D) Analogous to C, for the second studied configuration. The residue numbers are indicated, while the corresponding amino acid types are color-coded for both panels (C and D). (E and F) Time evolution of the fibronectin and arginine interaction energy with graphene for the two configurations. The lower plots in both panels show the fraction of arginine residue binding energy with respect to the total fibronectin-binding energy as a function of simulation time.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Binding Assay, Residue

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Mechanical properties. The measured quasi-static (A and B) and dynamic (C – E) properties of GF (hatched bars), GF coated in fibronectin (dark blue bars), and GF coated in fibronectin and cultured with ATDC5 cells (light blue bars) for 28 days. Fibronectin changed the elasticity of the composite (i.e., modulus values), but did not increase the viscoelastic properties (stress relaxation and phase shift).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Culture

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Actin cytoskeleton of cells on GF and fibronectin-coated GF. Fluorescence of ATDC5 cells grown on glass-bottom tissue culture wells compared to GF, with or without fibronectin. Cell nuclei are stained blue (DAPI); Green, F-actin (Alexa Fluor 488 phalloidin); (A–D) ATDC5 cells were grown on glass-bottom tissue culture wells without (A and E) and with fibronectin (B and F); ATDC5 cells were grown on GF without (C and G) and with fibronectin (D and H). Note the prevalence of stress fibers and the absence of puncta in F and H compared to E and G, respectively. Additionally, note the relative abundance of puncta of actin which are more prevalent in the absence of fibronectin on glass-bottomed tissue culture wells as well as on GF. (A–D) Scale-bar: 50 μm. (E–H) Scale-bar: 10 μm.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Fluorescence, Staining

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ActB and Hsp90ab1 housekeeping genes. ActB and Hsp90ab1 are stably expressed by ATDC5 cells under all experimental conditions used in this study (i.e., on glass-bottom tissue culture wells, GF, and fibronectin-GF). (A) ActB and Hsp90ab1 cycle threshold levels were most consistent among all samples analyzed by qRT-PCR for candidate HKGs considered, based on pairwise analysis of variance for differences between threshold values, variance equal to 0.12. (B) Correlation analysis of cycle threshold values for Hsp90ab1 and ActB indicate a slope and an R 2 value close to 1. ( n = 15).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Stable Transfection, Quantitative RT-PCR

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: GF supports or enhances gene expression levels. The effect of fibronectin, GF, and fibronectin in combination with GF on ATDC5 cell gene expression was investigated. Correlation analysis of relative expression levels was carried out to detect differential gene expression as a function of the cell culture substrate. The mRNA levels were compared for cells seeded on four distinct surfaces. Data points above the diagonal line indicate genes that are upregulated and data points below the diagonal line indicate genes that are downregulated. Data points falling on the diagonal line are not differentially expressed in experimental compared to control conditions. The effect of GF on gene expression is demonstrated in panels A and B. The effect of fibronectin on gene expression is demonstrated in panels C and D. (A) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the absence of fibronectin. (B) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the presence of fibronectin. (C) Relative gene expression levels in 2D cell culture conditions comparing the presence and absence of fibronectin. (D) Relative gene expression levels by cells grown in 3D on GF comparing the presence and absence of fibronectin. Genes for which expression levels met or exceeded the control are indicated in magenta, while those genes that were supported by substrate conditions are indicated by turquoise. Col2a1, a marker for chondrocyte differentiation, is shown as a diamond shape and bolded in each frame. Col2a1 is found above the diagonal line in A and B indicating upregulation as a function of 3D GF culture, and below the line in C and D, indicating downregulation as a function of fibronectin in either 2D or 3D culture. Genes included in this analysis are listed in Tables – .

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Gene Expression, Expressing, Cell Culture, Control, Marker

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding mediators of cell attachment by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Ctnnal (triangle) and Ctnnb1 (circle). (B) Relative gene expression levels of Ctnnal (gray) and Ctnnb1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Cd44 (triangle), Ncam1 (circle), and Sgce (square). (D) Relative gene expression levels of Cd44 (gray), Ncam1 (black), and Sgce (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Itga3 (triangle), Itga5 (circle), and Itgav (square). (F) Relative gene expression levels of Itga3 (gray), Itga5 (black ) , and Itgav (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Itgb1 . (H) Relative gene expression levels of Itgb1 at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD. These genes are listed in Table with references from current literature indicating an association with chondrocyte differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Cell Attachment Assay, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding extracellular matrix proteins by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Col1a1 (circle) and Col3a1 (triangle). (B) Relative gene expression levels of Col1a1 (gray) and Col3a1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Col2a1 (circle), Col5a1 (triangle), and Col6a1 (square). (D) Relative gene expression levels of Col2a1 (gray), Col5a1 (black), and Col6a1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Ecm1 (circle), Emilin1 (triangle), and Tnc (square). (F) Relative gene expression levels of Ecm1 (gray), Emilin1 (black), and Tnc (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Fn (circle), Sparc (triangle), and Spp1 (square). (H) Relative gene expression levels of Fn (gray), Sparc (black), and Spp1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (I) Time course of gene expression during chondrogenic differentiation for Thbs1 (circle), Thbs2 (triangle), and Postn (square). (J) Relative gene expression levels of Thbs1 (black), Thbs2 (white), and Postn (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (K) Time course of gene expression during chondrogenic differentiation for Hapln1 (circle) and Lamb3 (triangle). (L) Relative gene expression levels of Hapln1 (gray) and Lamb3 (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists extracellular matrix genes with description, function, and literature citations that corroborate an upregulation during early chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding matrix remodeling proteins and their endogenous inhibitors by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Adamts1 (circle) and Adamts2 (triangle). (B) Relative gene expression levels of Adamts1 (gray) and Adamts2 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Mmp2 (triangle) and Mmp14 (circle). (D) Relative gene expression levels of Mmp2 (black) and Mmp14 (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Timp1 (circle), Timp2 (triangle), and Timp3 (square). (F) Relative gene expression levels of Timp1 (gray), Timp2 (black), and Timp3 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Ctgf (circle) and Tgfbi (triangle). (H) Relative gene expression levels of Ctgf (gray) and Tgfbi (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists matrix remodeling genes analyzed in this study with descriptions and literature citations that have demonstrated a link between increases in gene expression and chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ECM Genes Expressed during Chondroprogenitor Cell Differentiation on GF

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Differentiation, Binding Assay, Activity Assay, Membrane

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 4. Stimulation of IVD cells with HTRA1-generated fibronectin fragments. A, concentrated protein supernatants (15 g) from IVD cells treated for 24 h without or with HTRA1mac (5 g/ml) or HTRA1macSA (5 g/ml) were subjected to immunoblotting using antibody Mab1935 specific for the fibronectincarboxyl-terminalheparin-bindingdomain(Cterminus)orMab1936specificforthefibronectinamino-terminalfibrin-andheparin-bindingdomain (N terminus). Fibronectin fragments containing the amino-terminal fibrin- and heparin-binding domain are identified by the closed arrowhead. B, purified human plasma-derived fibronectin (Fn) was incubated with HTRA1mac or HTRA1macSA at equimolar concentrations in TBS, pH 8.5, for 16 h at 37 °C, and samples were loaded onto a 4–15% gradient gel and stained with Coomassie Blue. Fibronectin and recombinant HTRA1 alone were also loaded and served as controls. C, an equimolar concentration of human plasma-derived fibronectin and HTRA1mac were incubated for 16 h, and fibronectin fragments were visualized by Western blot analysis using the antibodies described in A. D, equimolar concentrations of fibronectin (20 g) and HTRA1mac (5 g) were incubated for 16 h, and fibronectin fragments were purified by affinity chromatography. IVD cells were incubated with purified HTRA1-digested fibronectin (FnHTRA1mac) for 24 h, and expression levels of MMP1, MMP3, and MMP13 mRNA were determined by qRT-PCR and the -fold change as compared with untreatedcontrolswasdeterminedusingthe2CTmethod.Additionalcultureswereincubatedwitheitheraffinity-purifiedTris-bufferedsaline,pH7.6(TBS), fibronectin (Fn), or HTRA1 (HTRA1mac) or left untreated (Control). Data are representative of two separate experiments performed using IVD cells from two patients. Shown are results of triplicate determinations S.D. *, p 0.01, as determined by one-way ANOVA.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Generated, Western Blot, Binding Assay, Purification, Clinical Proteomics, Derivative Assay, Incubation, Staining, Recombinant, Concentration Assay, Affinity Chromatography, Expressing, Quantitative RT-PCR, Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 5. Detection of fibronectin fragments in degenerated IVD tissue. A, fibronectin (FN) mRNA levels in intact IVD tissue samples from patients (n 36) with varying degrees of IVD degeneration were determined by qRT-PCR and presented as 2CT S.E. (error bars). B, correlation study between FN and HTRA1 mRNA levels (2CT) in patient IVD tissue samples (n 36). R2, square of correlation coefficient; p 0.01 as determined from Pearson’s correlation coefficient. C, protein extracts from patient IVD tissues (n 12) were loaded onto a 12% SDS-polyacrylamide gel, and immunoblotting was performed using a monoclonal antibody (Mab1936) specific for the amino-terminal fibrin- and heparin-binding domain. D, the PVDF membrane used in C was stained with Coomassie Blue in order to confirm equal protein loading. Lane 1, HTRA1-digested human plasma-derived fibronectin; lanes 2–4, non-degenerated (ND) discs; lanes 5–7, mildly degenerated discs; lanes 8–10, moderately degenerated discs; lanes 11–13, severely degenerated discs.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Membrane, Staining, Clinical Proteomics, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 6. A theoretical model for the role of HTRA1 in IVD degeneration. Based on our findings, we propose that HTRA1 accumulates in IVD tissue undergoing degeneration and stimulates MMP production by resident cells in a predominantly protease-dependent manner, via activation of the MEK pathway. Furthermore, we suggest that the stimulatory effects of HTRA1 on IVD cells are mediated indirectly through its ability to generate fibronectin fragments, although other routes of cellular activation cannot be ruled out. IDD, intervertebral disc degeneration.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Activation Assay