Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: Integrin expression profiles are altered after OC-2 cell differentiation . (A) Western blot analysis of myosin VI and myosin VIIa levels in OC-2 cells grown at 33°C and 39°C. Both myosin VI and VIIa levels were increased significantly in cells grown at 39°C cells in comparison with those incubated at 33°C. Bar graphs represent densitometric results of mean relative values of myosin VI and myosin VIIa levels ± s.e.m. HSC-70 provided the loading control. (B) Undifferentiated (33°C) and differentiated (39°C) OC-2 cells were analysed by FACS for the integrin subunits α6, β1, αv and β3. Levels of β1-, αv- and β3-integrin subunits were increased significantly in differentiated OC-2 cells when compared with undifferentiated OC-2 cells. α6-integrin surface expressions levels did not change between the two cell phenotypes. Bar graph represents mean fluorescence units of the various integrin subunits ± s.e.m.; n = 3 independent experiments. White bars = cells at 33°C, black bars = cells at 39°C cells; nd = no significant difference, * P < 0.05, ** P < 0.01.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing, Cell Differentiation, Western Blot, Incubation, Fluorescence

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: Integrin expression profiles in the Organ of Corti . RT-PCR of RNA isolated from organ of Corti of adult mice. Bands at the predicted molecular weight are visible for integrin β1, β3, αv, α6 and β-actin but not for integrin β4. cDNA was omitted for the negative control.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Molecular Weight, Negative Control

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: αv-integrin expression profiles change during differentiation . Expression of (A) α6-integrin and (B) αv-integrin were examined by FACS at days 2, 6, 10 and 14 during the differentiation process. The differentiating cells ('dif' red line) were compared to undifferentiated ('33' green line) and fully differentiated cells ('39' red line). α6-integrin expression profiles did not change throughout the time course. αv-integrin expression profiles show an increase at day 6 and are at comparable levels with fully differentiated OC-2 cells at day 14 with an apparent even higher level at day 10.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: β1 and β3-integrin expression profiles change during differentiation . Surface expression of (A) β1-integrin and (B) β3-integrin was examined by FACS at days 2, 6, 10 and 14 of the differentiation process The differentiating cells ('dif' red line) were compared to undifferentiated ('33' green line) and fully differentiated cells ('39' red line). Increase of β1-integrin expression profile is visible from day 2 and it reaches the level of differentiated cells by day 10. β3-integrin expression which is undetectable above the Ig signal up to day 2, becomes visible at day 6 and gradually reaches the differentiated levels at day 14.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: β3-integrin is a marker for OC-2 cell differentiation . (A) Western blot analysis of myosin VIIa expression in 39°C OC-2 cells untreated or after treatment with either scrambled (Scr), β1-, α6-, αv- and β3-integrin siRNA. Treatment with all siRNAs reduced myosin VIIa levels significantly when compared with untreated OC-2 cells or OC-2 cells treated with scrambled siRNA. (B) Undifferentiated OC-2 cells transduced with human β3-integrin (+hβ3) had similar levels of surface β3-integrin to differentiated (39) OC-2 cells. Furthermore, analysis of the expression of myosin VIIa showed it was increased significantly in OC-2 cells transduced with human β3-integrin (+hβ3) cells compared with undifferentiated (33) cells. Bar graphs represent densitometric results of mean relative values of myosin VIIa levels ± s.e.m. HSC-70 was used as a loading control. * P < 0.05, ** P < 0.01. n = 3 individual experiments.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Marker, Cell Differentiation, Western Blot, Expressing, Transduction

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: Effect of β3-integrin-siRNA treatment on integrin surface expression . Differentiated OC2 cells were treated with β3-siRNA and assayed by FACS for expression of surface integrins. The data is compared to integrin expression levels in normal differentiated cells (39°), differentiated cells exposed to scrambled siRNA sequences (siRNAβ3) and undifferentiated cells (33°). Exposure to β3-integrin-siRNA resulted in a significant reduction of the surface expression of β3, β1- and αv- to levels similar to those of undifferentiated cells. There was no significant effect of scrambled siRNA sequences on any integrin expression and β3-integrin-siRNA did not significantly affect α6-integrin providing further evidence for the specificity of the siRNA inhibition.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing, Inhibition

Journal: BMC Cell Biology

Article Title: β3-integrin is required for differentiation in OC-2 cells derived from mammalian embryonic inner ear

doi: 10.1186/1471-2121-13-5

Figure Lengend Snippet: Graph of changes in expression levels of integrin subunits and myosin VIIa during OC-2 cell differentiation . Schematic time course graph showing fluorescence values of αv-, α6-, β1- and β3- integrin subunit surface expression measured by FACS and chemiluminescence values of the hair cell marker myosin VIIa in OC-2 cells measured by Western blot, over the 14 day differentiation process.

Article Snippet: Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK).

Techniques: Expressing, Cell Differentiation, Fluorescence, Marker, Western Blot

Journal:

Article Title: Dissection of platelet and myeloid cell defects by conditional targeting of the ?3-integrin subunit

doi: 10.1096/fj.09-138420

Figure Lengend Snippet: Successful disruption of the β3-integrin gene selectively in mouse platelets or OCs. A) β3-Integrin protein expression was absent in β3KOP mice. Representative Western blots of total β3-integrin protein in platelet lysates from 4- to 6-wk-old WT (first lane) and β3KOP mice (second lane). Total β-actin protein as loading control. B) β3-Integrin protein expression was absent during OC differentiation in β3KOM mice. Representative Western blots of total β3-integrin protein in OC lysates from WT and β3KOM mice at d 5 and 6 of OC differentiation. Lysates from d 6 β3−/− (lane 5) and β3+/+ (last lane) OCs were used as a control. Total β-actin protein as loading control. C) Absence of β3-integrin mRNA in lung endothelial cells (ECs) of WT mice. Endpoint RT-PCR was performed after EC isolation from pooled lung tissue (n=5) after a CD31 magnetic bead-mediated selection procedure. GAPDH was used as a housekeeping control gene. TEK, EC-specific receptor tyrosine kinase (∼200 bp); CO, control (no RT); LA, ladder. D) Detection of CD31 (red) and neighboring β3-integrin (green) in blood vessels recruited into a matrigel plug 10 d after subcutaneous placement of matrigel mixed with VEGF and heparin in β3KOP, β3KOM, and WT mice. Visualization by immunofluorescence. Nuclei are stained with DAPI. E) Number of vessel sprouts from aortic rings explanted from WT, β3KOP, β3KOM, and β3−/− mice and cultured in matrigel in the presence of VEGF. **P < 0.01 for β3−/− vs. WT mice. Data are means ± se.

Article Snippet: Frozen sections were then stained with rabbit anti-mouse polyclonal CD31 antibody (Abcam, Cambridge, MA, USA) and hamster anti-mouse monoclonal antibody against β3-integrin (BD Pharmingen, San Jose, CA, USA) and visualized by immunofluorescence.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Selection, Immunofluorescence, Staining, Cell Culture

Journal:

Article Title: Dissection of platelet and myeloid cell defects by conditional targeting of the ?3-integrin subunit

doi: 10.1096/fj.09-138420

Figure Lengend Snippet: Disruption of β3-integrin in platelets and megakaryocytes results in a bleeding phenotype. A) No differences observed in red blood cell (RBC), white blood cell (WBC), or platelet counts in β3KOP (solid bars) vs. TS-HET (hatched bars) and WT mice (open bars). B) Increased gastrointestinal bleeding events in β3KOP mice. Fecal occult blood tests were performed on feces samples from β3KOP and littermate controls and demonstrated a significantly higher number of positive tests in β3KOP mice. *P < 0.01 vs. controls; χ2 test. C) Increased bleeding times in β3KOP mice. Each symbol represents bleeding time on a single mouse according to genotype. In each case. bleeding in β3KOP mice had to be stopped by the examiner when bleeding did not stop spontaneously. P < 0.001; ANOVA with appropriate post hoc test. D, E) Platelet aggregation was dysfunctional in β3KOP platelets. D) In vivo clot retraction was assessed using pooled PRP from β3KOP mice and HET control mice. Red blood cells were added to enhance the color contrast for the photograph. E) Platelet aggregation assays on a slide (SPAT) were performed using PRP. No significant platelet aggregation was observed in PRP isolated from β3KOP mice; each test performed ≥3 times/genotype.

Article Snippet: Frozen sections were then stained with rabbit anti-mouse polyclonal CD31 antibody (Abcam, Cambridge, MA, USA) and hamster anti-mouse monoclonal antibody against β3-integrin (BD Pharmingen, San Jose, CA, USA) and visualized by immunofluorescence.

Techniques: In Vivo, Isolation

Journal:

Article Title: Dissection of platelet and myeloid cell defects by conditional targeting of the ?3-integrin subunit

doi: 10.1096/fj.09-138420

Figure Lengend Snippet: β3KOP mice have normal bone mass and osteoclast function. A) Expression of Pf4 in OC results in a reduction of β3-integrin mRNA in OCs from β3KOP mice compared to OC from WT mice. RT-PCR was performed using mRNA harvested from OCs of β3KOP and WT littermates at d 1–3 of OC differentiation. A ∼300 bp band shows abundance of β3-integrin message in WT OCs and a distinct level of β3-integrin expression in OCs from β3KOP mice. LA, marker; CO, negative control. GAPDH was used as a housekeeping control gene. B) β3-Integrin protein expression is reduced, but not absent, in β3KOP OCs at d 6 of OC differentiation compared to WT OCs. β3-Integrin protein from d 6 OC from β3−/− mice served as negative control and β-actin as loading control. C) OCs from WT, HET, and β3KOP mice demonstrated equivalent differentiation from bone marrow macrophages at d 2, 4, and 6 of OC differentiation. Representative TRAP staining of cultured macrophages and in vitro differentiated OCs from β3KOP mice and controls. D–F) BMD (D; n=6/group), serum CTX levels (E; n=10/group), and trabecular bone volume (F; n≥6/group) were equivalent in 6-mo-old WT (open bars), HET (hatched bars), and β3KOP mice (solid bars). G) Representative histology of femurs analyzed for trabecular bone volume.

Article Snippet: Frozen sections were then stained with rabbit anti-mouse polyclonal CD31 antibody (Abcam, Cambridge, MA, USA) and hamster anti-mouse monoclonal antibody against β3-integrin (BD Pharmingen, San Jose, CA, USA) and visualized by immunofluorescence.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control, Staining, Cell Culture, In Vitro

Journal:

Article Title: Dissection of platelet and myeloid cell defects by conditional targeting of the ?3-integrin subunit

doi: 10.1096/fj.09-138420

Figure Lengend Snippet: Myeloid-specific ablation of the β3-integrin gene recapitulates the osteopetrotic phenotype described in β3−/− mice. A, B) β3KOM mice (solid bars) demonstrated increased BMD compared to WT mice (open bars) at 2 mo (A) and at 6 mo of age (B). C) Trabecular bone volume was significantly increased in β3KOM mice vs. controls. D) Representative histology of femurs analyzed for trabecular bone volume (measured in C). Note the pronounced increase in trabecular bone in the β3KOM sample. E) CTX was significantly decreased in the serum of β3KOM mice vs. WT mice. F) Macrophages harvested and cultured from β3KOM mice demonstrated markedly dysfunctional differentiation into OCs on d 2–6 of OC differentiation compared to WT macrophages, as shown by representative in vitro TRAP staining. n ≥ 6/group. *P < 0.05; **P < 0.01.

Article Snippet: Frozen sections were then stained with rabbit anti-mouse polyclonal CD31 antibody (Abcam, Cambridge, MA, USA) and hamster anti-mouse monoclonal antibody against β3-integrin (BD Pharmingen, San Jose, CA, USA) and visualized by immunofluorescence.

Techniques: Cell Culture, In Vitro, Staining

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: β1 integrin-dependent adhesion of chondrocytes to CHAD. Culture dishes (48 well) were coated overnight with chondroadherin (5 μg/ml) and blocked for nonspecific binding with BSA (0.25%). Bovine chondrocytes were allowed to adhere to the dishes for 1 h at 37°C in the presence of various concentrations of a polyclonal antibody against the rat β1 integrin subunit or control IgG. Nonadherent cells were removed by washing, and adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control, and the numbers represent mean of duplicate adhesion from one of three experiments.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Binding Assay, Control

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Affinity purification of CHAD-binding cell surface proteins. Bovine chondrocytes were 125 I-labeled and lysed with 1% Triton X-100, 100 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM PMSF, 1 mM MnCl 2 , and 1 mM MgCl 2 in 10 mM Tris-HCl, pH 7.4. The lysate was passed over control agarose followed by CHAD agarose. Proteins with affinity for CHAD were eluted by EDTA (20 mM), passed over a desalting column (PD-10) equilibrated, and eluted with 0.3 M NaCl, 1% Triton X-100, 0.1% BSA 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM PMSF, in 50 mM Tris-HCl, pH 7.4. An aliquot of the protein peak was immunoprecipitated with the polyclonal rat β1 integrin antibody. Proteins in the eluate ( E ) and in the immunoprecipitate (β1) were separated by 4–12% SDS-PAGE under reducing ( R ) or nonreducing ( NR ) conditions.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Affinity Purification, Binding Assay, Labeling, Control, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Immunoprecipitation of CHAD-binding integrins from human fibroblasts. 125 I-labeled A549 fibroblasts were lysed with 1% Triton X-100, 100 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM PMSF, 1 mM MnCl 2 , and 1 mM MgCl 2 in 10 mM Tris-HCl, pH 7.4. The lysate was passed over control agarose followed by CHAD agarose. Proteins with affinity for CHAD were eluted by EDTA (20 mM), passed over a desalting column (PD-10) equilibrated, and eluted with 0.3 M NaCl, 1% Triton X-100, 0.1% BSA, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM PMSF in 50 mM Tris-HCl, pH 7.4. Aliquots of the protein peak were immunoprecipitated with monoclonal antibodies against the integrin subunits β1 (P4C10), α1 (TS2/7), α2 (P1E6), α5 (P1D6), and αv (VNR147). The immunoprecipitated proteins were separated by SDS-PAGE (4–12%) under nonreducing conditions and visualized by autoradiography.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Immunoprecipitation, Binding Assay, Labeling, Control, Bioprocessing, SDS Page, Autoradiography

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Immunoprecipitation of integrins from human fibroblasts. 125 I-labeled A549 fibroblasts were lysed with 1% Triton X-100, 100 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM PMSF, 1 mM MnCl 2 , and 1 mM MgCl 2 in 10 mM Tris-HCl, pH 7.4. Aliquots of the lysate were immunoprecipitated with monoclonal antibodies against the integrin subunits β1 (P4C10), β3 (RUU-PLF12), α1 (TS2/7), α2 (P1E6), α3 (P1B5), α5 (P1D6), αv (VNR147), and αvβ5 (P1F6). The immunoprecipitated proteins were separated by SDS-PAGE (4–12%) under nonreducing conditions and visualized by autoradiography.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Immunoprecipitation, Labeling, Bioprocessing, SDS Page, Autoradiography

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Inhibition of fibroblast adhesion to CHAD by integrin antibodies. Culture dishes (48 well) were coated with CHAD (5 μg/ml) and blocked for nonspecific binding with BSA (0.25%). Human A549 fibroblasts were allowed to adhere to the dishes for 1 h at 37°C in the presence of monoclonal antibodies against the human integrin subunits β1 (P4C10), β3 (RUU-PLF12), α2 (Gi9), α3 (P1B5), α5 (P1D6), αv (VNR147), αvβ3 (LM609), and αvβ5 (P1F6). Nonadherent cells were removed by washing, and adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control, and the numbers represent the mean of duplicate adhesion from three individual experiments ±SD. * P < 0.05; ** P < 0.01; P β1 = 0.002; P α2 = 0.011; P α3 = 0.021.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Inhibition, Binding Assay, Bioprocessing, Control

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Inhibition of human chondrocyte adhesion to CHAD by α2 integrin antibodies. Culture dishes (48 well) were coated with CHAD (5 μg/ml) and blocked for nonspecific binding with BSA (0.25%). Human chondrocytes were allowed to adhere to the dishes for 1 h at 37°C in the presence of various concentrations of the monoclonal antibody against the human integrin subunit α2 (Gi9). Nonadherent cells were removed by washing, and adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control, and the numbers represent the mean adhesion ±SD from three wells in one of two experiments.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Inhibition, Binding Assay, Control

Journal: The Journal of Cell Biology

Article Title: Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

doi:

Figure Lengend Snippet: Inhibition of adhesion of T47D cells to CHAD ( a ) and to collagen type II (CII; b ) by various α2 antibodies. Culture dishes (48 wells) were coated with 5 μg/ml of CHAD or CII and blocked for nonspecific binding with BSA (0.25%). T47D cells were allowed to adhere to the dishes for 1 h at 37°C in the absence or in the presence of monoclonal antibodies against the integrin subunits β1 (P4C10), β3 (RUU-PLF12), or various α2 antibodies. Nonadherent cells were removed by washing, and adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control, and the numbers represent the mean of duplicate adhesion from three individual experiments ±SD. * P < 0.05; ** P < 0.01; ( a ) P β1 = 0.004; P Gi9 = 0.006; P Gi19 = 0.026. ( b ) P β1 = 0.000; P P1E6 = 0.001; P P1H5 = 0.001; P Gi9 = 0.000; P Gi26 = 0.047.

Article Snippet: Monoclonal antibody against the human integrin β3 (RUU-PLF12, purified IgG) were purchased from Becton Dickinson (Bedford, MA).

Techniques: Inhibition, Binding Assay, Bioprocessing, Control