Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Expression of signaling molecules associated with cellular invasiveness including integrins and Rho GTPases under hypoxia. (a) Protein expression levels of integrins and phosphorylated FAK under normoxic or hypoxic conditions. (b) The expression levels of RhoA, ROCK1, Rac1/2/3, and phosphorylated Rac1/cdc42. GAPDH was used as the loading control. ∗ P < 0.05 (compared with normoxic group). ITGA4: integrin α 4 ; ITGA5: integrin α 5 ; ITGB7: integrin β 7 ; p-FAK: phosphorylated focal adhesion kinase; pRac1/cdc42: phosphorylated Rac1/cdc42.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Alteration of integrin α 4 -mediated signaling pathway in BM-MSCs under hypoxia. The mRNA expression levels of HIF-1 α (a) and integrin α 4 (b) in BM-MSCs were determined by real-time PCR. 18S rRNA was used as the loading control. Protein expression levels of integrin α 4 (c) and ROCK1 and Rac1/2/3 (d) in BM-MSCs were assessed by Western blotting. GAPDH was used as the loading control. ∗ P < 0.05 (compared with YC-1 nontreated group) and # P < 0.05 (compared with normoxic group). HIF-1 α : hypoxia-inducible factor-1 α ; ITGA4: integrin α 4 ; ROCK1: Rho-associated kinase 1.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: The effect of integrin α 4 inhibition on BM-MSC migration and activities of MMPs under hypoxia. (a) The mRNA expression of integrin α 4 in BM-MSCs was suppressed by transfection of integrin α 4 siRNA. 18S rRNA was used as the loading control. (b) BM-MSC migration was significantly increased after siITGA4 transfection. Invasiveness of BM-MSCs was assessed by invasion assay (left). BM-MSCs invaded through the inserts were counted for quantification (right). (c) Enzymatic activities of MMP-9 and MMP-2 in BM-MSCs after siITGA4 transfection were determined by zymography (left). Quantification of enzymatic activities of MMP-9 (middle) and MMP-2 (right). ∗ P < 0.05 (compared with siITGA4 nontransfected group) and # P < 0.05 (compared with normoxic group). MMP: matrix metalloproteinase; siITGA4: integrin α 4 siRNA.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Inhibition, Migration, Expressing, Transfection, Invasion Assay, Zymography

Journal: Stem Cells International

Article Title: Hypoxia Inducible Factor-1 α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α 4

doi: 10.1155/2016/7932185

Figure Lengend Snippet: Interaction between integrin α 4 and HIF-1 α and its effect on expression of Rho GTPases under hypoxia. (a) Protein expression levels of integrin α 4 , HIF-1 α , ROCK1, and Rac1/2/3 were assessed by Western blotting. GAPDH was used as the loading control. (b) HIF-1 α and integrin α 4 were localized with immunofluorescence in BM-MSCs after siITGA4 transfection under normoxic or hypoxic conditions. Blue: DAPI; green: HIF-1 α ; red: integrin α 4 . Scale bar = 80 μ m (400x original magnification). ∗ P < 0.05 (compared with siITGA4 nontransfected group) and # P < 0.05 (compared with normoxic group). DAPI: 4′,6-diamidino-2-phenylindole; HIF-1 α : hypoxia-inducible factor-1 α ; ITGA4: integrin α 4 ; ROCK1: Rho-associated kinase 1; siITGA4: integrin α 4 siRNA.

Article Snippet: The following primary antibodies were used: rabbit anti-HIF-1 α (1 : 1,000, BD Biosciences, San Jose, CA, USA), rabbit anti-integrin α 4 (1 : 1,000, ProSci-Inc., Poway, CA, USA), rabbit anti-integrin α 5 (1 : 1,000, BD Biosciences), mouse anti-integrin β 7 (1 : 1,000, R&D Systems, Minneapolis, MN, USA), rabbit anti-RhoA (1 : 1,000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ROCK1 (1 : 1,000, Cell Signaling Technology), rabbit anti-Rac1/2/3 (1 : 2,000, Cell Signaling Technology), rabbit antiphosphorylated Rac1/cdc42 (1 : 1,000, Cell Signaling Technology), and rabbit antiphosphorylated focal adhesion kinase (FAK) (1 : 500, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection

Journal: Cell Death & Disease

Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

doi: 10.1038/cddis.2015.50

Figure Lengend Snippet: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P <0.01, compared with GFP vector). Protein expression was confirmed by western blotting assays using GFP-tagged antibody when equal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference protein. ( b and c ) RUFY3 colocalizes with F-actin at the cell periphery. SGC-7901 cells were transiently transfected with pEGFP-C1 or pEGFP-RUFY3. Rhodamine-conjugated phalloidin was used to detect F-actin. After 24 h transfection, cells were fixed and permeabilized. ( b ) Images were captured using a scanning confocal fluorescence microscope and one confocal section is shown in each image. Scale bars, 10 μ m. ( c ) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with F-actin at the cell periphery. The data show mean±S.E.M. (** P <0.01, compared with GFP vector), in which ~40 transfected cells were observed. ( d ) Colocalization of GFP-RIPX and myosinIIb at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of myosinIIb (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 10 μ m. ( e ) Colocalization of GFP-RIPX and integrin β 5 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RIPX. Colocalization of integrin β 5 (red) with GFP-RIPX is shown by yellow fluorescence. Scale bars, 20 μ m. ( f ) Colocalization of GFP-RIPX and integrin α 3 β 1 at the cell periphery by plating cells on vitronectin is observed by confocal microscopy. Scale bars, 20 μ m

Article Snippet: The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) for 3 h at room temperature, and the proteins were probed with specific antibodies: GFP and His (GenScript Corporation, Nanjing, China), Flag (Shanghai Kangcheng), PAK1, integrin β 5 and myosinIIb (Cell Signaling), RUFY3 and vinculin (Santa Cruz), integrin α 3 β 1 (Bioss Inc. and Abcam).

Techniques: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Expressing, Western Blot, Fluorescence, Microscopy, Confocal Microscopy

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces the migration of MSCs partly through the FAK/STAT3 signaling pathway. (A) Transwell assays for the migration of MSCs using 152RM (n = 5 each). Representative crystal violet staining images are shown in the left panel. Quantification of cell migration is shown in the right panel. Scale bar, 100 μm. (B) RNA-seq analysis showed the alteration of cell migration-specific gene expression in MSCs cultured with 152RM (n = 3 each). (C) Relative mRNA expression levels of cell migration-specific genes in MSCs cultured with 152RM (n = 5 each). (D) Quantification of the transwell assay and cell wound scratch assay after culture with 152RM, a CXCR4 inhibitor (AMD3100) and an Integrin αvβ3 inhibitor (Cyclo(-RGDfK)) (n = 5 each). (E) Western blot analysis of the expression of E-cadherin in MSCs after the addition of 152RM (n = 5 per group). (F) Gene set enrichment analysis (GSEA) plots showing upregulation of the JAK/STAT signaling pathway in MSCs cultured with 152RM (n = 3 each). (G) RNA-seq analysis showed alterations in JAK/STAT signaling pathway-related gene expression in MSCs cultured with 152RM (n = 3 each). (H) Western blot analysis of the expression of CXCR4, integrin αvβ3, p-Jak2, Jak2, p-FAK, FAK, p-STAT3 and STAT3 in MSCs (pretreated with a CXCR4 inhibitor (AMD3100) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Staining, RNA Sequencing Assay, Expressing, Cell Culture, Transwell Assay, Wound Healing Assay, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: 152RM induces ECs migration partly through the FAK/ERK signaling pathway. (A) Transwell assay for the migration of ECs using 152RM (n = 5 each). Scale bar, 100 μm. Quantification of cell migration was performed (right). (B) Relative mRNA expression levels of cell migration-specific genes in ECs cultured with 152RM (n = 5 each). (C) Representative immunostaining images of p-VEGFR2 (red) ECs with or without 152RM (n = 5 per group). Scale bar, 100 μm. (D) Quantification of transwell assays after culture with 152RM, a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor cyclo(-RGDfK) (n = 5 each). (E) Western blot analysis of the expression of integrin αvβ3, p-VEGFR2, p-FAK, FAK, p-ERK1/2 and ERK1/2 in ECs (pretreated with a VEGFR2 inhibitor (Ki8751) and an integrin αvβ3 inhibitor (cyclo(-RGDfK))) after the addition of 152RM (n = 5 per group). (F) Schematic illustration of the role of 152RM in promoting ECs migration. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA and Student's t -test were employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: Migration, Transwell Assay, Expressing, Cell Culture, Immunostaining, Western Blot

Journal: Bioactive Materials

Article Title: Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering

doi: 10.1016/j.bioactmat.2020.12.025

Figure Lengend Snippet: DBM-MSN/152RM scaffolds coordinate the recruitment of MSCs and ECs in vivo . (A) HE staining images demonstrating the recruitment of MSCs and ECs 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (B) Co-immunofluorescence staining of CXCR4, integrin αvβ3 and CD271 in MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (C) Immunofluorescence staining of CD90 + CD105 + MSCs from 1 week after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (D) Co-immunofluorescence staining of emcn and integrin αvβ3 in type H vessels from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. (E) Immunofluorescence staining of CD31 + in EPCs from 4 weeks after DBM, DBM-MSN, DBM/152RM and DBM-MSN/152RM scaffold implantation (n = 5 rats per group). Scale bar, 100 μm. Data are shown as the mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA was employed. For all panels in this figure, data are representative of three independent experiments.

Article Snippet: Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C.

Techniques: In Vivo, Staining, Immunofluorescence

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) Chemical structure of DOTA-cycratide. (B) Inhibition of 64Cu-cycratide binding to integrin αvβ6 on BxPC-3 cells by cycratide, DOTA-cycratide, and linear-pep. Data are shown as mean ± SD, n = 4. (C) Binding of 68Ga-cycratide to BxPC-3 with or without blocking of cold cycratide or linear-pep. %AD/106 cells = percentage of total added dose per million cells. Data are shown as mean ± SD, n = 4. (D) Metabolic stability of 68Ga-cycratide in blood and urine of BALB/c mice (data are representative of 3 independent experiments). **P < 0.01.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Inhibition, Binding Assay, Blocking Assay

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET imaging of orthotopic pancreatic cancer lesions in nude mice at 0.5 h after injection of 68Ga-cycratide without or with blocking dose of cold cycratide. Tumors are indicated by arrows. (B) Hematoxylin and eosin (H&E) staining of tumor tissues harvested from orthotopic tumor model. (C) Left, immunofluorescence staining of integrin αvβ6 from tumor tissues harvested from orthotopic tumor model. Right, negative control with secondary antibody only.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Imaging, Injection, Blocking Assay, Staining, Immunofluorescence, Negative Control

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET/CT images of female patient with suspected pancreatic cancer. Images were obtained at 1 h after intravenous administration of 68Ga-cycratide or 18F-FDG. Tumors are indicated by arrows. (B) Immunohistochemical (IHC) staining for integrin αvβ6 in tumor sample from same patient as in A. (C) Contrast-enhanced CT (CECT) image and PET/CT images of male patient with pancreatic cancer 7 mo after surgery at 1 h after administration of 68Ga-cycratide or 18F-FDG, as well as CECT image of same patient 3 mo later (10 mo after surgery). Occupancy lesions in CT are indicated by arrows.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Positron Emission Tomography-Computed Tomography, Immunohistochemical staining, Immunohistochemistry

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: In situ binding of recombinant integrins to frozen sections of mouse embryonic tissues. (A–C) Recombinant integrin α8β1 (green) bound to E15.5 dorsal skin (A), lung (B), and choroid plexus (C) cryosections. The top and bottom panels show fluorescence images for wild-type and Qbrick −/− littermates, respectively. (D–F) Integrins α3β1 (D), α6β4 (E), and α7X2β1 (F) bound to dorsal skin cryosections from wild-type (top) and Qbrick −/− (bottom) littermates were visualized (green). (G and H) Integrin α8β1 (green) bound to dorsal skin cryosections from Frem2 my /+ and Frem2 my / my (G) and Grip1 eb /+ and Grip1 eb / eb (H) mice. The top and bottom panels show fluorescence images for control heterozygotes and homozygotes, respectively. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Immunoreactivity against QBRICK (green) is also shown. Arrowheads indicate the location of BMs. The asterisks indicate nonspecific binding of recombinant integrins or the anti-QBRICK antibody to the cornified epithelium. Bars, 20 µm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: In Situ, Binding Assay, Recombinant, Fluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired binding of integrin α8β1 in the developing kidney of Qbrick −/− mice. (A) Unilateral renal agenesis observed in Qbrick −/− mice. Bar, 5 mm. (B and C) The left panels show integrin α8β1 (green) bound to the BMs of the E10.5 mesonephric duct (B) and E11.5 ureteric bud (C) of wild-type (top) and Qbrick −/− (bottom) embryos. The right panels show the BMs counterstained with an anti–laminin-γ1 chain antibody (magenta). (D and E) QBRICK immunofluorescence (green) in the developing kidney of wild-type (top) and Qbrick −/− (bottom) embryos at E10.5 (D) and E11.5 (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). The open and closed arrowheads indicate the mesonephric duct and ureteric bud, respectively. The asterisks indicate nonspecific binding of the anti-QBRICK antibody to the mesenchyme. Bars, 50 µm. (F) The expression levels of Gdnf and Pax2 transcripts in the E11.5 metanephros of Qbrick +/− control (open bars) and Qbrick −/− (shaded bars) littermates were determined by quantitative RT-PCR and normalized by the expression level of Gapdh . The expression level in control mice was set at 1. Each bar represents the mean ± SD (error bars; n = 3). **, P < 0.01, significant difference by Student’s t test.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Immunofluorescence, Expressing, Control, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Generation of Qbrick RGE/RGE mice. (A) Schematic representation of the targeted mutation of Qbrick . Open boxes represent exons. The knock-in construct was designed to replace exon 4, which contains the codon encoding Asp207, with a mutated exon (open box with asterisk) in which the codon encoding Asp207 was substituted with a codon encoding Glu207. The probes used for Southern blotting are indicated by bold lines. N, NcoI restriction site; TK, thymidine kinase. (B) Direct DNA sequencing of the wild-type and Qbrick RGE/RGE genomes. The numbers indicate the positions of the amino acids in the primary sequence of QBRICK protein. (C) Southern blot analyses of genomic DNA from wild-type, Qbrick RGEneo/+ , and Qbrick RGE/+ offspring after digestion with NcoI. Fragments of 5.3 and 7.7 kbp are detected with probe 1 and probe 2, respectively, indicating that the expected homologous recombination occurred. The detection of a 6.1-kbp fragment with probe 2 indicates that the neomycin resistance gene has been removed from the Qbrick RGEneo allele by the Cre-loxP system. (D and E) Immunofluorescence staining (green) for QBRICK in E15.5 dorsal skin (D) and E11.5 ureteric buds (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). (F–H) Qbrick RGE/RGE mice develop normally without any defects in the kidneys (asterisks in F), eyelids (G), and digits (H). (I and J) Binding of recombinant integrin α8β1 (green) to E15.5 dorsal skin (I) and E11.5 ureteric buds (J). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In panels D, E, I, and J, the top and bottom panels show fluorescence images for wild-type and Qbrick RGE/RGE littermates, respectively. The expression levels of QBRICK are comparable between wild-type and Qbrick RGE/RGE littermates. It should be noted that the binding of integrin α8β1 to the epidermal BMs of the E15.5 dorsal skin (I) as well as the E11.5 ureteric buds (J) is comparable between wild-type and Qbrick RGE/RGE littermates. Bars: (D and I) 20 µm; (E and J) 50 µm; (F) 5 mm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Mutagenesis, Knock-In, Construct, Southern Blot, DNA Sequencing, Sequencing, Homologous Recombination, Immunofluorescence, Staining, Binding Assay, Recombinant, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired expression of nephronectin and MAEG in FS model mice. (A–F) Immunofluorescence staining (green) for nephronectin (A, C, and E) and MAEG (B, D, and F) in the dorsal skin of Qbrick −/− (A and B), Frem2 my / my (C and D), and Grip1 eb / eb (E and F) mice, and their control wild-type or heterozygous littermates. (G–J) Immunofluorescence staining (green) for nephronectin (G and H) and MAEG (I and J) at the E10.5 mesonephric duct (G and I; open arrowheads) and E11.5 ureteric bud (H and J; closed arrowheads) in wild-type (top) and Qbrick −/− (bottom) littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Bars: (A–F) 20 µm; (G–J) 50 µm. (K and L) Immunoblot detection of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), nephronectin (Npnt), and MAEG in protein extracts from the kidney (K) and skin (L) of E15.5 Qbrick −/− mice and their wild-type littermates. (M–O) Immunoblot signal intensities of laminin, integrin α8, nephronectin, and MAEG in the kidney (left) and skin (right) of E15.5 wild-type (open bars) and Qbrick −/− (shaded bars; M), E15.5 Frem2 my /+ (open bars) and Frem2 my / my (shaded bars; N), and E17.5 Grip1 eb /+ (open bars) and Grip1 eb / eb (shaded bars; O) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant differences by Student’s t tests. (P) Titration curves of recombinant integrin α8β1 bound to the GST-fused NV domain of QBRICK (open triangles), GST-fused RGD linker segment of nephronectin (open diamonds), GST-fused RGD linker segment of MAEG (open squares), and GST (closed circles). Each point represents the mean ± SEM ( n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Titration, Recombinant

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: The binding of integrin α8β1 to BMs is not compromised in Maeg ΔRGD mice. (A) Schematic views of MAEG and MAEG ΔRGD , a deletion mutant lacking the linker segment. The sites recognized by the anti-MAEG antibodies (Anti-linker and Anti-MAM) are also shown. (B) Schematic representation of the targeted mutation of Maeg . Open boxes represent exons. The targeting construct was designed to replace exons 9 and 10, which encode the linker region, with PGKneo. The probes used for Southern blotting are indicated by bold lines. B, BamHI restriction site; TK, thymidine kinase. (C) Southern blot analysis of genomic DNA from wild-type, Maeg ΔRGD/+ , and Maeg ΔRGD/Y mice digested with BamHI. The detection of a 6.3-kbp fragment instead of an 8.2-kbp fragment indicates the presence of the mutated allele. (D) Direct DNA sequencing of the Maeg ΔRGD/Y -derived RT-PCR product demonstrating that the open reading frame has not shifted. The product encodes a polypeptide lacking amino acids 258–391 of the 550–amino-acid full-length sequence. The boundary between exons 8 and 11 is shown by the red broken line. The numbers indicate the positions of the corresponding amino acids in the wild-type MAEG sequence. (E and F) In E15.5 dorsal skin epidermal BMs, MAEG is comparably detected by immunofluorescence (green) in both Maeg +/Y and Maeg ΔRGD/Y littermates with the anti-MAM antibody (E), but is only detected in Maeg +/Y embryos by the anti-linker antibody (F). (G, H, and K) Immunofluorescence staining for QBRICK (G, green) and nephronectin (H, green), and binding of recombinant integrin α8β1 (K, green) to epidermal BMs of the E15.5 dorsal skin are comparable between Maeg +/Y and Maeg ΔRGD/Y littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In each set of panels, the top and bottom panels show fluorescence images for wild-type Maeg +/Y and Maeg ΔRGD/Y littermates, respectively. Bars, 20 µm. (I and J) Immunoblot signal intensities of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), and nephronectin (Npnt) in the kidney (I) and skin (J) of E15.5 control Maeg +/Y (open bars) and Maeg ΔRGD/Y (shaded bars) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Mutagenesis, Construct, Southern Blot, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Immunofluorescence, Staining, Recombinant, Fluorescence, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: A model for renal defects in FS. In the developing metanephros, QBRICK and other FS-associated proteins associate with nephronectin in the sublamina densa region and secure its localization at BMs. Metanephric mesenchymal cells interact with nephronectin through integrin α8β1. Stimulation of integrin α8β1 induces Gdnf , which is necessary for further development of the metanephros (left). In FS, nephronectin is not stably deposited at BMs, owing to the loss of FS-associated proteins, and is eventually degraded. The loss of nephronectin leads to failure of Gdnf induction and subsequent metanephric development (right).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Stable Transfection