7A). polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30C60 min, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting exhibited proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. = 34) with allergy and/or asthma by unfavorable selection using a cocktail of anti-CD16, anti-CD14, anti-CD3, and anti-glycophorin beads as before [11, 12, 32]. The purity and viability of eosinophils were 98%. The studies were approved by the University of Wisconsin-Madison Health Sciences Institutional Review Board. Informed written consent was obtained from each subject before participation. Periostin and ADAM8 The shortest carboxy (C)-terminal splice variant of human periostin, i.e., lacking sequences encoded by differentially spliced exons 17, 18, 19, and 21 (PN0, UniProt identifier No. “type”:”entrez-protein”,”attrs”:”text”:”Q15063″,”term_id”:”93138709″,”term_text”:”Q15063″Q15063C7), was cloned into pAcGP67.coco (hereafter pCOCO-PN0), expressed in insect cells using a baculovirus system, and purified as previously described [12, 33, 34]. The complementary DNA (cDNA) sequences corresponding to periostin FAS1 1C2 (residues P97-L365 relative to M1 of PN0), FAS1 2 (G234-L365), FAS1 2C3 (G234-I492), FAS1 3C4 (D368-L628), and FAS1 3 module-C terminus (D368-Q721) were amplified by polymerase chain reaction (PCR). The periostin FAS1 3-C terminus cDNA sequence was amplified from the pCOCO-PN0 plasmid, thus lacking alternative exons 17, 18, 19, and 21. Primers were designed to obtain PCR amplicons with 5 (1,200 rpm in a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). Supernatants were carefully aspirated (leaving 50 l), precipitated in glass tubes with 80% acetone at -20C overnight, and centrifuged for 10 min at 4C at 8,000 (8,500 rpm in an SS-34 rotor, Sorvall RC-5B centrifuge, Du Pont). Cell pellets and supernatant precipitates were resuspended in 25 l PBS, then dissolved by adding 50 l 4% SDS, 4 M urea, 5% glycerol, 62.5 mM Tris, pH 6.8 with bromophenol blue, i.e., to a total volume of 75 l (corresponding to 10 106 cells). Samples (15 l per lane) were run under non-reducing conditions on 8% SDS-PAGE. Thus, each Rabbit Polyclonal to GAK 15 l (each lane of) cell or supernatant sample contained material originating from 2 106 cells. Immunoblotting was performed by transfer to polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo mini PVDF Transfer Pack and Bio-Rad Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA) (for periostin constructs) or as described [34] (for cell and supernatant samples), incubation with primary antibodies at 0.5 g/ml, and detection of bands by peroxidase-conjugated secondary antibodies at 1:20,000 and enhanced chemiluminescence (SuperSignal? West Pico Chemiluminescent Substrate, Thermo Scientific, Madison, WI, USA, or Perkin Elmer, Waltham, MA, USA, respectively). Specificity of the secondary antibodies was assessed by omitting the primary antibodies. Images were processed using the BioSpectrum 810 imaging system and VisionWorks LS software (UVP, Upland, CA, USA). Cell motility assay Cell motility was assessed as before [12] with the following modifications. Wells were coated with 10 g/ml periostin from R&D, blocked with fetal bovine serum (FBS). Then 1 m-diameter Polybeads were added to the wells [12]. After eosinophils were resuspended at 2 106/ml in RPMI with 20% FBS, they were allowed to rest for 1 h at 37C. Eosinophils were then diluted 1:100 to 20,000/ml in RPMI-20% FBS and 50.Podosomes in tumor cells are associated with local loss and degradation of the ECM ligand, e.g., fibronectin and collagen, immediately under the podosome [39]. The Stiny-1 mAb epitope, which we map here to the FAS1 4 module of periostin, was lost from the periostin layer over large areas in the vicinity of eosinophils. a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. = 34) with allergy and/or asthma by negative selection using a cocktail of anti-CD16, anti-CD14, anti-CD3, and anti-glycophorin beads as before [11, 12, 32]. The purity and viability of eosinophils were 98%. The studies were approved by the University of Wisconsin-Madison Health Sciences Institutional Review Board. Informed written consent was obtained from each subject before participation. Periostin and ADAM8 The shortest carboxy (C)-terminal splice variant of human periostin, i.e., lacking sequences encoded by differentially spliced exons 17, 18, 19, and 21 (PN0, UniProt identifier No. “type”:”entrez-protein”,”attrs”:”text”:”Q15063″,”term_id”:”93138709″,”term_text”:”Q15063″Q15063C7), was cloned into pAcGP67.coco (hereafter pCOCO-PN0), expressed in insect cells using a baculovirus system, and purified as previously described [12, 33, 34]. The complementary DNA (cDNA) sequences corresponding to periostin FAS1 1C2 (residues P97-L365 relative to M1 of PN0), FAS1 2 (G234-L365), FAS1 2C3 (G234-I492), FAS1 3C4 (D368-L628), and FAS1 3 module-C terminus (D368-Q721) were amplified by polymerase chain reaction (PCR). The periostin FAS1 3-C terminus cDNA sequence was amplified from the pCOCO-PN0 plasmid, thus lacking alternative exons 17, 18, 19, and 21. Primers were designed to obtain PCR amplicons with 5 (1,200 rpm in a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). Supernatants were carefully aspirated (leaving 50 l), precipitated in glass tubes with 80% acetone at -20C overnight, and centrifuged for 10 min at 4C at 8,000 (8,500 rpm in an SS-34 rotor, Sorvall RC-5B centrifuge, Du Pont). Cell pellets and supernatant precipitates were resuspended in 25 l PBS, then dissolved by adding 50 l 4% SDS, 4 M urea, 5% glycerol, 62.5 mM Tris, pH 6.8 with bromophenol blue, i.e., to a total volume of 75 l (corresponding to 10 106 cells). Samples (15 l per lane) were run under non-reducing conditions on 8% SDS-PAGE. Thus, each 15 l (each lane of) cell or supernatant sample contained material originating from 2 106 cells. Immunoblotting was performed by transfer to polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo mini PVDF Transfer Pack and Bio-Rad Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA) (for periostin constructs) or as described [34] (for cell and supernatant samples), incubation with primary antibodies at 0.5 g/ml, and detection of bands by peroxidase-conjugated secondary antibodies at 1:20,000 and enhanced chemiluminescence (SuperSignal? West Pico Chemiluminescent Substrate, Thermo Scientific, Madison, WI, USA, or Perkin Elmer, Waltham, MA, USA, respectively). Specificity of the secondary antibodies was assessed by omitting the primary antibodies. Images were processed using the BioSpectrum 810 imaging system and VisionWorks LS software (UVP, Upland, CA, USA). Cell motility assay Cell motility was assessed as before [12] with the following modifications. Wells were coated with 10 g/ml periostin from R&D, blocked with fetal bovine serum (FBS). Then 1 m-diameter Polybeads were added to the wells [12]. After eosinophils were resuspended at 2 106/ml in RPMI with 20% FBS, they were allowed to rest for 1 h at 37C. Eosinophils were then diluted 1:100 to 20,000/ml in RPMI-20% FBS and 50 l (i.e., 1,000 cells) was added to each well (containing a bead layer in 50 l RPMI) in the presence of IL-5 (10 ng/ml final concentration) and incubated for 20 h at 37C. Wells.(I-L) Quantitation of changes in morphology and F-actin and PSGL-1 localization in eosinophils adherent to periostin in the presence of IL-5 for 10, 30 or 60 min, using the Fiji version of ImageJ (http://fiji.sc/Fiji): (I) cell circumference, (J) cell area, (K) peripheral F-actin staining as percentage of circumference, and (L) peripheral PSGL-1 staining as percentage of circumference (mean SEM, = 30 cells at each time point, * 0.05, *** 0.001 versus 10 min). The morphology of the punctate structures in eosinophils adhered to periostin in the presence of IL-5, as noted briefly previously [12], characterizes podosomes, dynamic adhesive contacts distinct from classical focal adhesions and present in many cell types, including cancer cells, macrophages, osteoclasts, dendritic cells, and vascular smooth muscle cells. in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30C60 min, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. = 34) with allergy and/or asthma by negative selection using a cocktail of anti-CD16, anti-CD14, anti-CD3, and anti-glycophorin beads as before [11, UF010 12, 32]. The purity and viability of eosinophils were 98%. The studies were authorized by the University or college of Wisconsin-Madison Health Sciences Institutional Review Table. Informed written consent was from each subject before participation. Periostin and ADAM8 The shortest carboxy (C)-terminal splice variant of human being periostin, i.e., lacking sequences encoded by differentially spliced exons 17, 18, 19, and 21 (PN0, UniProt identifier No. “type”:”entrez-protein”,”attrs”:”text”:”Q15063″,”term_id”:”93138709″,”term_text”:”Q15063″Q15063C7), was cloned into pAcGP67.coco (hereafter pCOCO-PN0), expressed in insect cells using a baculovirus system, and purified while previously described [12, 33, 34]. The complementary DNA (cDNA) sequences related to periostin FAS1 1C2 (residues P97-L365 relative to M1 of PN0), FAS1 2 (G234-L365), FAS1 2C3 (G234-I492), FAS1 3C4 (D368-L628), and FAS1 3 module-C terminus (D368-Q721) were amplified by polymerase chain reaction (PCR). The periostin FAS1 3-C terminus cDNA sequence was amplified from your pCOCO-PN0 plasmid, therefore lacking alternate exons 17, 18, 19, and 21. Primers were designed to obtain PCR amplicons with 5 (1,200 rpm inside a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). Supernatants were cautiously aspirated (leaving 50 l), precipitated in glass tubes with 80% acetone at -20C over night, and centrifuged for 10 min at 4C at 8,000 (8,500 rpm in an SS-34 rotor, Sorvall RC-5B centrifuge, Du Pont). Cell pellets and supernatant precipitates were resuspended in 25 l PBS, then dissolved by adding 50 l 4% SDS, 4 M urea, 5% glycerol, 62.5 mM Tris, pH 6.8 with bromophenol blue, i.e., to a total volume of 75 l (related to 10 106 cells). Samples (15 l per lane) were run under non-reducing conditions on 8% SDS-PAGE. Therefore, each 15 l (each lane of) cell or supernatant sample contained material originating from 2 106 cells. Immunoblotting was performed by transfer to polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo mini PVDF Transfer Pack and Bio-Rad Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA) (for periostin constructs) or as explained [34] (for cell and supernatant samples), incubation with main antibodies at 0.5 g/ml, and detection of bands by peroxidase-conjugated secondary antibodies at 1:20,000 and enhanced chemiluminescence (SuperSignal? Western Pico Chemiluminescent Substrate, Thermo Scientific, Madison, WI, USA, or Perkin Elmer, Waltham, MA, USA, respectively). Specificity of the secondary antibodies was assessed by omitting the primary antibodies. Images were processed using the BioSpectrum 810 imaging system and VisionWorks LS software (UVP, Upland, CA, USA). Cell motility assay Cell motility was assessed as before [12] with the following modifications. Wells were coated with 10 g/ml periostin from R&D, clogged with fetal bovine serum (FBS). Then 1 m-diameter Polybeads were added to the wells [12]. After eosinophils were resuspended at 2 106/ml in RPMI with 20% FBS, they were allowed to rest for 1 h at 37C. Eosinophils were then diluted 1:100 to 20,000/ml in RPMI-20% FBS and 50 l (i.e., 1,000 cells) was added to each well (comprising a bead coating in 50 l RPMI) in the presence of IL-5 (10 ng/ml final concentration) and incubated for 20 h at 37C. Wells were viewed in an Eclipse Ti inverted microscope (Nikon Tools). Images were acquired and exported using NIS-Elements AR. Results were quantified using Fiji. Statistical analysis Combined 0.05 was considered significant. Analyses were performed using Prism (GraphPad Inc., San Diego, CA, USA). Results Upon adhesion to periostin in the presence of IL-5, eosinophils transition from a polarized.Images were acquired and exported using NIS-Elements AR. (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in additional cell types. Instead, immunoblotting shown proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-triggered eosinophils on periostin show loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, consequently, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. = 34) with allergy and/or asthma by bad selection using a cocktail of anti-CD16, anti-CD14, anti-CD3, and anti-glycophorin beads as before [11, 12, 32]. The purity and viability of eosinophils were 98%. The studies were authorized by the University or college UF010 of Wisconsin-Madison Health Sciences Institutional Review Table. Informed written consent was obtained from each subject before participation. Periostin and ADAM8 The shortest carboxy (C)-terminal splice variant of human periostin, i.e., lacking sequences encoded by differentially spliced exons 17, 18, 19, and 21 (PN0, UniProt identifier No. “type”:”entrez-protein”,”attrs”:”text”:”Q15063″,”term_id”:”93138709″,”term_text”:”Q15063″Q15063C7), was cloned into pAcGP67.coco (hereafter pCOCO-PN0), expressed in insect cells using a baculovirus system, and purified as previously described [12, 33, 34]. The complementary DNA (cDNA) sequences corresponding to periostin FAS1 1C2 (residues P97-L365 relative to M1 of PN0), FAS1 2 (G234-L365), FAS1 2C3 (G234-I492), FAS1 3C4 (D368-L628), and FAS1 3 module-C terminus (D368-Q721) were amplified by polymerase chain reaction (PCR). The periostin FAS1 3-C terminus cDNA sequence was amplified from your pCOCO-PN0 plasmid, thus lacking alternate exons 17, 18, 19, and 21. Primers were designed to obtain PCR amplicons with 5 (1,200 rpm in a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). Supernatants were cautiously aspirated (leaving 50 l), precipitated in glass tubes with 80% acetone at -20C overnight, and centrifuged for 10 min at 4C at 8,000 (8,500 rpm in an SS-34 rotor, Sorvall RC-5B centrifuge, Du Pont). Cell pellets and supernatant precipitates were resuspended in 25 l PBS, then dissolved by adding 50 l 4% SDS, 4 M urea, 5% glycerol, 62.5 mM Tris, pH 6.8 with bromophenol blue, i.e., to a total volume of 75 l (corresponding to 10 106 cells). Samples (15 l per lane) were run under non-reducing conditions on 8% SDS-PAGE. Thus, each 15 l (each lane of) cell or supernatant sample contained material originating from 2 106 cells. Immunoblotting was performed by transfer to polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo mini PVDF Transfer Pack and Bio-Rad Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA) (for periostin constructs) or as explained [34] (for cell and supernatant samples), incubation with main antibodies at 0.5 UF010 g/ml, and detection of bands by peroxidase-conjugated secondary antibodies at 1:20,000 and enhanced chemiluminescence (SuperSignal? West Pico Chemiluminescent Substrate, Thermo Scientific, Madison, WI, USA, or Perkin Elmer, Waltham, MA, USA, respectively). Specificity of the secondary antibodies was assessed by omitting the primary antibodies. Images were processed using the BioSpectrum 810 imaging system and VisionWorks LS software (UVP, Upland, CA, USA). Cell motility assay Cell motility was assessed as before [12] with the following modifications. Wells were coated with 10 g/ml periostin from R&D, blocked with fetal bovine serum (FBS). Then 1 m-diameter Polybeads were added to the wells [12]. After.Primers were designed to obtain PCR amplicons with 5 (1,200 rpm in a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). end. After 30C60 min, the nucleopod experienced dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting exhibited proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target. = 34) with allergy and/or asthma by unfavorable selection using a cocktail of anti-CD16, anti-CD14, anti-CD3, and anti-glycophorin beads as before [11, 12, 32]. The purity and viability of eosinophils were 98%. The studies were approved by the University or college of Wisconsin-Madison Health Sciences Institutional Review Table. UF010 Informed written consent was obtained from each subject before participation. Periostin and ADAM8 The shortest carboxy (C)-terminal splice variant of human periostin, i.e., lacking sequences encoded by differentially spliced exons 17, 18, 19, and 21 (PN0, UniProt identifier No. “type”:”entrez-protein”,”attrs”:”text”:”Q15063″,”term_id”:”93138709″,”term_text”:”Q15063″Q15063C7), was cloned into pAcGP67.coco (hereafter pCOCO-PN0), expressed in insect cells using a baculovirus system, and purified as previously described [12, 33, 34]. The complementary DNA (cDNA) sequences corresponding to periostin FAS1 1C2 (residues P97-L365 relative to M1 of PN0), FAS1 2 (G234-L365), FAS1 2C3 (G234-I492), FAS1 3C4 (D368-L628), and FAS1 3 module-C terminus (D368-Q721) were amplified by polymerase chain reaction (PCR). The periostin FAS1 3-C terminus cDNA sequence was amplified from your pCOCO-PN0 plasmid, thus lacking alternate exons 17, 18, 19, and 21. Primers were designed to obtain PCR amplicons with 5 (1,200 rpm in UF010 a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE, USA). Supernatants were cautiously aspirated (leaving 50 l), precipitated in glass tubes with 80% acetone at -20C overnight, and centrifuged for 10 min at 4C at 8,000 (8,500 rpm in an SS-34 rotor, Sorvall RC-5B centrifuge, Du Pont). Cell pellets and supernatant precipitates were resuspended in 25 l PBS, then dissolved by adding 50 l 4% SDS, 4 M urea, 5% glycerol, 62.5 mM Tris, pH 6.8 with bromophenol blue, i.e., to a total volume of 75 l (corresponding to 10 106 cells). Samples (15 l per lane) were run under nonreducing circumstances on 8% SDS-PAGE. Therefore, each 15 l (each street of) cell or supernatant test contained material from 2 106 cells. Immunoblotting was performed by transfer to polyvinylidene difluoride (PVDF) membranes using Bio-Rad Trans-Blot Turbo mini PVDF Transfer Pack and Bio-Rad Trans-Blot Turbo Blotting Program (Bio-Rad, Hercules, CA, USA) (for periostin constructs) or as referred to [34] (for cell and supernatant examples), incubation with major antibodies at 0.5 g/ml, and detection of bands by peroxidase-conjugated secondary antibodies at 1:20,000 and improved chemiluminescence (SuperSignal? Western Pico Chemiluminescent Substrate, Thermo Scientific, Madison, WI, USA, or Perkin Elmer, Waltham, MA, USA, respectively). Specificity from the supplementary antibodies was evaluated by omitting the principal antibodies. Images had been prepared using the BioSpectrum 810 imaging program and VisionWorks LS software program (UVP, Upland, CA, USA). Cell motility assay Cell motility was evaluated as before [12] with the next modifications. Wells had been covered with 10 g/ml periostin from R&D, clogged with fetal bovine serum.