Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 1. Inhibition of fibronectin expression in UUO kidney by blockade of IL-17A. (a ∼d). IL-17A (a & b) and fibronectin expression (c & d) in 7-day post-UUO kidney (b & d) and unobstructed contralateral kidney (control) (a & c) was determined by immunohistochemistry. Faint staining of IL-17A was observed in normal tubular cells of the control kidney, whereas strong staining IL-17A was found in tubular cells of UUO kidney. Fig. 1, Panel a represents relative percentage of IL-17A staining using immunohistochemistry (IHC) of Fig. 1a and b. Fig. 1, Panel b represents relative percentage of fibronectin staining using IHC of Fig. 1c and d. One represented section is demonstrated in figures (a–d). Magnification: a, b, c, d: ×400.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Inhibition, Expressing, Control, Immunohistochemistry, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 2. Decreased fibronectin expression, ERK1/2 activation, and TGF-β1 expression by blockade of IL-17A function. Mice received UUO for 7 days and extracellular matrix in kidney specimens was stained with Masson's Trichrome (a: the contralateral control kidney; b: the UUO kidney). Mice were injected in- travenously with 100 μg anti-IL-17A receptor antibody through the tail vein at 2 h before UUO and on days 1 and 3 after UUO (c). One represented section is demonstrated in figures. Fig. 2a, Panel a represents relative percentage of fibrosis staining using IHC of Fig. 2a, 2b, and 2c. Kidney parenchymal lysate was subjected to immunoblotting analysis to determine IL-17A and fibronectin expression (d) and p-ERK1/2 and total ERK1/2 levels (e). The density of each protein band was normalized with that of GAPDH, and relative fold changes compared to the control of Fig. 2d are displayed in the Fig. 2d, Panel a (white bars: fibronectin expression; black bars: IL-17A expression) and relative fold changes compared to the control of Fig. 2e are displayed in the Fig. 2e, Panel a. One representative result from three independent experiments is shown (*p < 0.05). TGF-β1 mRNA expression was analyzed by qPCR. TGF-β1 mRNA expression was normalized to GAPDH, and relative fold changes compared to the control are displayed (n = 4 in each group; *p < 0.05) (f). Kidney parenchymal lysate was subjected to immunoblotting analysis to determine TGF-β1 expression (g). Fig. 2g, Panel a represents western blot analysis shown in Fig. 2g. The groupings of gels were cropped from different parts of the same gel. Magnification: a, b, c: ×200.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Expressing, Activation Assay, Staining, Control, Injection, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 4. IL-17A–induced increase in fibronectin production. (a). HRPTECs were serum-deprived for 24 h and then grown in the presence or absence of different concentrations of IL-17A (5−100 ng/mL) under serum-free conditions for 2 days. Fig. 4a, Panel a represents wester blot analysis shown in Fig. 4a. (b). Cells were treated with 50 ng/mL IL-17A for dif- ferent periods, as indicated. Quantitative analysis of each protein band was displayed in the supplemental Fig. S2. Cell lysates were subjected to immunoblotting analysis for the measurement of fibronectin production. GAPDH expression was used as a loading control. One representative experiment from at least three independent experiments is shown. The groupings of gels were cropped from different parts of the same gel.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Western Blot, Expressing, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 5. Blockade of the IL-17A–stimulated increase of ERK1/2 activation and fibronectin expression by an ERK1/2 inhibitor. (a). HRPTECs were stimulated with IL-17A (50 ng/mL) under serum-free conditions for the designated periods. (b & c). Cells were pre-treated with different con- centrations of U0126 (2.5−10 μM/mL) in the presence or absence of IL-17A (50 ng/mL) under serum-free conditions for 7.5 min (b) or 48 h (c). ERK1/2 kinase activity was determined by immunoblotting with an anti-p-ERK1/2 antibody, and the same immunoblot membrane was probed again and total ERK1/2 proteins were then determined. GAPDH expression was used as a loading control. Quantitative analysis of these Western blot analyses was displayed in the supplemental Fig. S3∼5. The groupings of gels were cropped from different parts of the different gels of the same specimen.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Membrane, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 6. Inhibition of ERK1/2 activation blocks IL- 17A–induced ECM expression in renal fibroblasts. (a). Hs891 T cells were stimulated with different concentrations of IL-17A (5−100 ng/mL) and grown under serum-free condi- tions for 2 days. Cell lysates were subjected to immunoblotting to determine the expression of fibronectin and type I collagen. (b & c). Cells were pre-treated with different concentrations of U0126 (b: 5−10 μM/mL; c: 2.5−10 μM/mL) in the presence or absence of IL-17A (50 ng/mL) under serum-free conditions for 7.5 min (b) or 48 h (c). ERK1/2 kinase activity was de- termined by immunoblotting with an anti-p-ERK1/2 antibody, and the same immunoblot membrane was probed again and total ERK1/2 proteins were then determined. Fibronectin and type I collagen levels in the supernatant were determined by immunoblotting (c). Quantitative analysis of these Western blot analyses was displayed in the supplemental Figs. S6–S8. The groupings of gels were cropped from different parts of the different gels of the same specimen.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Activity Assay, Membrane

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 7. Inhibition of IL-17A–stimulated fibronectin using a neutralizing anti-TGF-β1 antibody. a. Cells were serum-deprived for 48 h and stimulated with IL-17A (50 ng/mL) for a further 30 min to 4 h (a). TGF-β1 mRNA ex- pression was analyzed by qPCR. TGF-β1 mRNA expression was normalized to GAPDH, and relative fold changes compared to the control are displayed (n = 5 in each group; *p < 0.05). (b). Cells were treated with an anti-TGF-β1 neu- tralizing antibody (100 ng/mL) in the presence or absence of IL-17A (50 ng/ mL) or TGF-β (5 ng/mL, positive control) for 48 h. (c). Cells were pretreated with different concentrations of an anti-TGF-β1 neutralizing antibody (10−100 ng/mL) followed by stimulation with IL-17A (50 ng/mL) for 48 h. Cell lysates were subjected to immunoblotting to determine fibronectin expression. Quantitative analysis of these Western blot analyses was displayed in the sup- plemental Fig. S9∼S10. The groupings of gels were cropped from different parts of the same gel.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Inhibition, Expressing, Control, Positive Control, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways.

doi: 10.1016/j.biopha.2019.109741

Figure Lengend Snippet: Fig. 8. Inhibition of IL-17A–induced fibronectin expression by inhibiting Smad2/3 activation. (a). Cells were stimulated with IL-17A (50 ng/mL) under serum- free conditions for different periods, as indicated. Cell lysates were subjected to immunoblotting to assess p-Smad2 and p-Smad3 levels. (b ∼d). Cells were pretreated with different concentrations of an anti-TGF-β1 neutralizing antibody (10−100 ng/mL) or SB431542 (5 or 10 μM/mL) followed by stimulation with IL-17A (50 ng/ mL) for 24 h. p-Smad2 or fibronectin levels were determined by immunoblotting. (e). Cells were treated with 50 nM/mL p-Smad2 or p-Smad3 siRNA overnight and incubated with 50 ng/mL IL-17A for 48 h. Fig. 8e, Panel a represents western blot analysis shown in Fig. 8e. Cell lysates were subjected to immunoblotting to determine Smad2, Smad3, and fibronectin levels. The presented results are representative of three independent experiments. Quantitative analysis of these Western blot analyses was displayed in the supplemental Fig. S11∼S14. The groupings of gels were cropped from different parts of the different gels of the same specimen.

Article Snippet: The catalog number of fibronectin antibody was MAB1918 (R&D Systems) with 1:100 dilutions.

Techniques: Inhibition, Expressing, Activation Assay, Western Blot, Incubation

Journal: Journal of Functional Biomaterials

Article Title: Multi-Composite Bioactive Osteogenic Sponges Featuring Mesenchymal Stem Cells, Platelet-Rich Plasma, Nanoporous Silicon Enclosures, and Peptide Amphiphiles for Rapid Bone Regeneration

doi: 10.3390/jfb2020039

Figure Lengend Snippet: Number of CB MSC attached to bacterial grade (non-adherent) plastic coated with various protein-containing solutions after 1 hour. 10% PRP resulted in the greatest cell retention, while both PRP and PPP were superior to gelatin or fibronectin coatings ( A ). Percentage of cells retained by coated or gelled PCL (gray) and collagen (white) scaffolds after 24 h compared to MSC seeded onto tissue culture plastic. PRP proved to be the most potent coating agent, while PRP and PRP-PA gels retained greater than 90% of loaded cells in PRP-coated collagen scaffolds ( B ). PA gels were significantly greater for cell retention than any of the coatings (δ, p = 3.0 × 10 −7 ). PA gels retained significantly less cells in either scaffold material than the PRP only gel (Ω, p = 0.012) or the PRP + PA gel carrier (Π, p < 0.002). Collagen retained significantly more cells than PCL scaffolds when delivered via PRP only gel (†, p = 0.023) or PRP + PA gel (‡, p = 0.010).

Article Snippet: Recombinant human bone morphogenetic protein-2 (BMP2) and recombinant human fibronectin were purchased from R&D Systems (Minneapolis, MN, U.S.).

Techniques:

Journal: eLife

Article Title: Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

doi: 10.7554/eLife.58688

Figure Lengend Snippet: Migration of activated PBT plated onto fresh tumor slices was analyzed in EGI-1 and MMTV-PyMT tumor model, whilst resident tumor-infiltrating T lymphocytes were analyzed in mPDAC and KPC tumor model. Illustrative images of T cell migration tracks in EGI-1, MMTV-PyMT, mPDAC, and KPC tumor models. Tumor stroma (fibronectin) in red, tumor cells (EpCAM in EGI-1, MMTV-PyMT, and KPC tumor models, CD44 in mPDAC tumor models), in blue and T cells (CD8 in mPDAC and KPC, Calcein in MMTV-PyMT, and EGI-1 tumor models) in green. Tracks are color-coded to illustrate track displacement. Scale bar = 100 µm. T cell migration speed, T cell displacement, and trajectory straightness in all tumor models. ***p - value>0.001, p-value>0.05, Student’s t-test. Results are shown as mean ± SD. Figure 5—source data 1. Source data file for .

Article Snippet: Live vibratome sections were stained with BV421 anti-mouse EpCAM (G8.8 clone; BD Biosciences), PerCP-e710 anti-mouse CD8a (53–6.7 clone, eBioscience), PE anti-podoplanin (8.1.1 clone; BioLegend), and anti-human/mouse fibronectin (HFN7.1 clone; Novus Biologicals).

Techniques: Migration

Journal: eLife

Article Title: Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

doi: 10.7554/eLife.58688

Figure Lengend Snippet: ( A ) Representative images of Control and BAPN-treated KPC tumors. Tumor cells (EpCAM) in blue, tumor stroma (fibronectin) in red, and T cells (CD8) in green. Scale bar = 100 µm. ( B ) Quantification of CD8 + T cells per mm 2 in tumor islets and stroma regions in control and BAPN (*p<0.01, Student’s t-test, n = 2–3 mice/group). Figure 5—figure supplement 1—source data 1. Source data file for .

Article Snippet: Live vibratome sections were stained with BV421 anti-mouse EpCAM (G8.8 clone; BD Biosciences), PerCP-e710 anti-mouse CD8a (53–6.7 clone, eBioscience), PE anti-podoplanin (8.1.1 clone; BioLegend), and anti-human/mouse fibronectin (HFN7.1 clone; Novus Biologicals).

Techniques: Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 1 Deregulation of extracellular matrix components after long-term CCM3 inactivation. A, Western Blot analyzes verified complete CCM3 inactivation in clonally expanded CCM3−/− CI-huVECs used in this study (clones I-IV). Expression levels normalized to the CCM3+/+ control group are given below the panel. B, RNA-Seq data of CCM3+/+ control (x-axis) and CCM3−/− CI-huVECs (y-axis) are presented as scatter dot plot. FPKM = fragments per kilobase of exon model per million mapped reads. C, Expression levels of FN1, FBLN5, and POSTN were validated by qPCR. D, Western Blot results revealed less fibronectin in cell culture supernatants of CCM3−/− CI-huVECs and reduced DOC-insoluble fibronectin aggregates upon CCM3-inactivation. Expression levels normalized to the CCM3+/+ control group are given below the subpanels. E, Fluorometric cell adhesion assays demonstrated no major cell binding abnormalities of CCM3−/− CI-huVECs to ECM components. F, A reduced fibronectin expression was observed in immunofluorescence imaging of 1 × 104 cells/well cultured on a tissue culture treated 96-well plate after 48 hours. Plasma fibronectin supplementation promoted fibronectin matrix assembly. Scale bars ≙ 100 µm in the left and 50 µm in the right panels. Images were acquired using the same setting for each sample and no changes were implemented. ND = not detected, RFU = relative fluorescence units, ctrl = CCM3+/+ control cells, Col I = type I collagen, Col II = type II collagen, Col IV = type IV collagen, FN = fibronectin, LN = laminin, TN = tenascin, VN = vitronectin, and Neg = bovine serum albumin. Data are presented as mean and single data points (n = 3-4). Multiple t tests were used for statistical analyzes. ****P < .0001

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Western Blot, Clone Assay, Expressing, Control, RNA Sequencing, Cell Culture, Binding Assay, Immunofluorescence, Imaging, Clinical Proteomics, Fluorescence

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 2 Restored endothelial function of CCM3−/− CI-huVECs by fibronectin replacement. A, CCM3−/− CI-huVECs cultured on fibronectin coated plates (5 µg/cm2) regained a typical endothelial morphology. Black arrowheads indicate a compact cell shape, while white arrowheads indicate cells that show a spindle-shaped morphology. CCM3+/+ and CCM3−/− CI-huVECs were seeded with 1 × 104 cells/well on a 96-well plate. Scale bar ≙ 200 µm. B, Fibronectin supplementation (32 µg/mL) significantly improved the spheroid organization of CCM3−/− CI-huVECs. The circularity and the cross-sectional area of the spheroids were determined. The manually traced perimeter of the shown spheroid is depicted in the upper right corner. Scale bar ≙ 100 µm. C, CCM3−/− CI-huVECs cultured on fibronectin-coated plates demonstrated a reduced actin stress fiber formation (1 × 104 cells/well; 96-well plate). Confocal microscopy was used for image acquisition. Phalloidin-iFluor 488 and DAPI staining are shown in green and blue, respectively. The brightness was adjusted equally for all images to show the relevant structures of F-actin formation. Original images are shown in Figure S1. Scale bar ≙ 50 µm. D, The reduced ability of CCM3−/− CI-huVECs to form tube- like structures could not be rescued by fibronectin supplementation (32 µg/mL). Scale bar ≙ 1 mm. E, Neither fibronectin coating (5 µg/cm2) nor supplementation to the culture medium (32 µg/mL) had an effect on staurosporine-induced Caspase-3 activity. F, Representative Phospho-Kinase array membranes are shown for CCM3−/− CI-huVECs cultured without and with fibronectin supplementation (5 µg/cm2). Spots showing the detection of phosphorylated forms of Src and FAK are marked in green or red, respectively. G and H, RNA-Seq data of CCM3+/+ control cells without (x-axis) and CCM3−/− CI-huVECs with 5 µg/cm2 fibronectin supplementation (y-axis) (G) or CCM3−/− CI-huVECs without (x-axis) and with (y-axis) fibronectin supplementation (H) are presented as scatter dot plot. FPKM = fragments per kilobase of exon model per million mapped reads. ctrl = CCM3+/+ control cells, FN = fibronectin, Nb = Number. Data are presented as mean and single data points (n = 3-5). Two-way ANOVA with Holm-Šidák's multiple comparisons test, multiple t test or Student's t test were used for statistical analyzes: *P < .05; **P < .01; ****P < .0001

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Cell Culture, Confocal Microscopy, Staining, Activity Assay, RNA Sequencing, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 3 Fibronectin replacement improves spheroid organization of CCM3−/− hCMEC/D3 cells. A, Western Blot analyzes verified complete CCM3 inactivation in clonally expanded hCMEC/D3 used in this study (clones I-III). Expression levels normalized to the CCM3+/+ control group are given below the panel. B, A reduced fibronectin expression was observed in immunofluorescence imaging of cells cultured on a tissue culture treated 96-well plate (1 × 104 cells/well). Scale bars ≙ 200 µm in the left and 50 µm in the right panels. C, Plasma fibronectin supplementation (32 µg/mL) significantly improved spheroid organization of CCM3−/− hCMEC/D3 cells. Shown are the circularity and the area of the spheroids. Scale bar ≙ 100 µm. ctrl = CCM3+/+ hCMEC/D3 cells, FN = fibronectin, ND = not detected. Data are presented as mean and single data points (n = 3). Student's t test was used for statistical analyzes: ***P < .001

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Western Blot, Clone Assay, Expressing, Control, Immunofluorescence, Imaging, Cell Culture, Clinical Proteomics

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 4 The 120 kD fragment of fibronectin is sufficient to rescue the cytoskeletal changes and spheroid organization of CCM3−/− CI-huVECs. A, Phalloidin-iFluor 488-(green) and DAPI-(blue) co-staining demonstrated that the actin stress fiber content was significantly decreased in CCM3−/− CI-huVECs that had been cultured on plates coated with cFN, 70 kD or 120 kD fibronectin fragments (5 µg/cm2; 1 × 104 cells/well). Scale bar ≙ 50 µm. B, Only the supplementation of a 120 kD fibronectin fragment (32 µg/mL) and type IV collagen (60 µg/mL) but not of cellular fibronectin (cFN) or a 70 kD fibronectin fragment (32 µg/mL) significantly rescued circularity and proper spheroid organization of CCM3−/− CI-huVECs. Scale bar ≙ 100 µm. ctrl = CCM3+/+ control cells, cFN = cellular fibronectin, 70 kD FN = 70 kD fibronectin fragment, 120 kD FN = 120 kD fibronectin fragment, Col IV = type IV collagen. Data are presented as mean and single data points (n = 3). Two-way ANOVA with Holm-Šidák's multiple comparisons test was used for statistical analyzes: ***P < .001, ****P < .0001

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Staining, Cell Culture, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 6 Deregulation of fibronectin expression upon CCM1 or CCM2 inactivation in CI-huVECs. A, High proportion of CCM1 and CCM2 loss-of-function alleles were found in crRNA:tracrRNA:Cas9 RNP-treated CI-huVEC mixtures. B, Immunofluorescence staining in crRNA:tracrRNA:Cas9 RNP-treated CI-huVECs indicated reduced fibronectin expression (1 × 104 cell/well, 96-well plate). Scale bars ≙ 200 µm in the left and 50 µm in the right panels. C, T7EI analyzes revealed no alterations in predicted off-target regions. Three independent replicates are shown (I-III). Expected length of uncleaved (black) and cleaved (gray) amplicons are depicted at the upper left of each subpanel. crRNA CCM1 = CCM1-targeting RNP, crRNA CCM2 = CCM2-targeting RNP, nc crRNA = non-targeting control RNP, FN = fibronectin. Data are presented as mean and single data points (n = 3)

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: The FASEB Journal

Article Title: Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3

doi: 10.1096/fj.201902888r

Figure Lengend Snippet: FIGURE 7 Fibronectin replacement attenuates endothelial dysfunction upon CCM1 or CCM2 inactivation in CI-huVECs. A, crRNA:tracrRNA:Cas9 RNP-treated CI-huVECs demonstrated a high proportion of cells with a compact morphology, which was significantly decreased by fibronectin supplementation (1 × 104 cell/well; 96-well plate). Black arrowheads indicate the compact cell shape while white arrowheads indicate cells that regained a spindle-shaped morphology after fibronectin supplementation. Scale bar ≙ 200 µm. B and C, CCM1 and CCM2 inactivation in crRNA:tracrRNA:Cas9 RNP-treated cell mixtures led to an increased stress fiber formation (1 × 104 cells/well; 96-well plate, (B) and an impaired spheroid organization (C), which both were attenuated by fibronectin supplementation. Scale bars ≙ 50 µm (B) and 100 µm (C). crRNA CCM1 = CCM1-targeting RNP, crRNA CCM2 = CCM2-targeting RNP, nc crRNA = non-targeting control RNP. Data are presented as mean and single data points (n = 3). Two-way ANOVA with Holm-Šidák's multiple comparisons test or Student's t test were used for statistical analysis: *P < .05; **P < .01; ***P < .001; ****P < .0001

Article Snippet: About 10 or 20 μg total protein were separated on 7.5% or 10% of TGX stain-free gels (Bio-Rad, Hercules, California, USA), transferred to PVDF membranes (Roche, Basel, Switzerland), immunostained with monoclonal mouse anti-human fibronectin (1:5000, MAB19182, R&D systems) or rabbit anti-CCM3 (1:150; IG-626, ImmunoGlobe, Himmelstadt, Germany), HRP conjugated mouse IgGκ light chain binding protein (1:30 000, sc-516102, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit HRP secondary antibody (1:30 000, sc2357, Santa Cruz Biotechnology), Precision Protein StrepTactin-HRP Conjugate (1:15 000, Bio-Rad) and detected with Clarity Western ECL (Bio-Rad).

Techniques: 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 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