Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Structure of human tenascin-C. A, Organization of the tenascin-C molecule. This diagram is adapted from Aukhil et al. (1993). The N termini of three arms are joined to form a trimer, and two trimers are connected via a disulfide bond (S-S) to form a hexamer. Each arm consists of 14 domains with homology to epidermal growth factor (EGF), 8-17 FN-III repeats depending on alternative RNA splicing, and a single fibrinogen (fbg) domain. The universal FN-III repeats (fn1-5 and fn6-8) are present in all tenascin-C splice variants. The largest tenascin-C splice variant contains nine alternatively spliced FN-III repeats (designated A1, A2, A3, A4, B, AD1, AD2, C, and D, or fnA-D), which are missing in the shortest splice variant. B, Crystal structure of universal tenascin-C FN-III domain 3 (fn3). This structure is adapted from Leahy et al. (1992) and consists of six exposed loops and seven β strands. The α9β1 integrin recognition sequence EIDGIELT, which corresponds to the neurite outgrowth-promoting sequence VFDNFVLK in fnD, is highlighted and includes portions of an exposed loop and adjacent β strand.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Variant Assay, Sequencing

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: A β1 integrin neuronal receptor is implicated in promotion of neurite outgrowth by fnD and VFDNFVLK. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound fnD or VFDNFVLK and a function-blocking antibody againstβ1 integrin subunit (10 μg/ml). Distributions of total neurite length are presented as a box-and-whisker plot. One representative experiment of four is shown. Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers indicate 5th and 95th percentiles. FnD and VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody did not alter neurite outgrowth on PLL but completely blocked outgrowth promotion by fnD and VFDNFVLK. B, Neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound plus excess soluble (bd + sl) VFDVFVLK and β1 integrin antibody. One representative experiment of three is shown. Outgrowth values were higher than those observed in A for neurons cultured on bound peptide alone and reduced to control values in the presence of β1 antibody.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay, Whisker Assay, Control

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Neurons cultured on bound VFDNFVLK but not VSPNGSLK demonstrate clustering of immunoreactivity for β1 integrin subunit. Immunocytochemistry was performed using a monoclonal hamster antibody against β1 integrin chain, followed by a fluorescein-conjugated goat anti-hamster secondary antibody. VFDNFVLK but not VSPNGSLK induced clustering of neuronal β1 integrin immunoreactivity. Scale bar, 10 μm.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Cerebellar granule neurons express the X2B isoform of the α7 integrin subunit. A, Homogenate from P8 rat cerebellar granule neurons (25 μg) was separated on 8% SDS-PAGE gels under nonreducing conditions and then transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody, which cross-reacts with all splice variants of the α7 integrin chain, or polyclonal α7A or α7B antibodies, which cross-react with variants containing the A or B cytoplasmic tail. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots probed with the O26 or α7B antibody demonstrated one predominant polypeptide band with Mr of 120,000, whereas blots probed with the α7A antibody revealed no polypeptide band. B, RT-PCR was performed on total RNA prepared from P8 rat cerebellar granule neurons. Amplification was done using sense and antisense primers designed to amplify X1, X2, A, and B variants of the α7 integrin as described in Materials and Methods. The 200 bp fragment corresponds to the X2 extracellular variant, and the 370 bp fragment corresponds to the B cytoplasmic variant. No products were obtained that corresponded to the X1 extracellular variant (220 bp) or the A cytoplasmic variant (480 bp).

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: SDS Page, Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: The α7 integrin subunit is implicated as the partner for β1 in promotion of neurite outgrowth by VFDNFVLK. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound VFDNFVLK and function-blocking O26 antibody against α7 integrin subunit (100, 50, 10, 5, 1, or 0.5 μg/ml). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The α7 integrin antibody did not alter neurite outgrowth on PLL at any concentration used. The antibody inhibited promotion of outgrowth by VFDNFVLK and demonstrated a biphasic concentration dependence. Outgrowth was significantly impaired at 50, 10, and 5 μg/ml O26 antibody (**p < 0.05; Kolmogorov-Smirnov test), with maximal inhibition at 10 μg/ml.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay, Control, Concentration Assay, Inhibition

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: The α1, α2, α3, and α6 integrin subunits do not play a role in VFDNFVLK-facilitated neurite growth. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound VFDNFVLK and function-blocking antibodies against α1, α2, or α3 integrin subunits (10 μg/ml) (A), whereas mouse cerebellar granule neurons were cultured in the presence of bound VFDNFVLK and a function-blocking antibody against α6 integrin subunit (10 μg/ml) (B). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). None of the antibodies altered neurite outgrowth on PLL or VFDNFVLK.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: α7 integrin is involved in neurite outgrowth promotion by tenascin-C and laminin-1 but not by fibronectin. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound large tenascin-C and function-blocking antibody O26 against α7 integrin subunit or function blocking antibody against β1 integrin subunit. One representative experiment of three is shown. Tenascin-C significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially blocked outgrowth promotion by large tenascin-C; the reduction in outgrowth values was significant (**p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody completely blocked outgrowth promotion by large tenascin-C. B, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound laminin-1 or fibronectin and function-blocking O26 antibody against α7 integrin subunit. One representative experiment of three is shown. Laminin-1 and fibronectin significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially reduced neurite outgrowth on laminin-1; the reduction was significant (**p < 0.05; Kolmogorov-Smirnov test). The antibody did not alter neurite outgrowth on fibronectin.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Neuronal α7β1 integrin binds to D5 peptide in an FD-FV-dependent manner. P8 cerebellar granule neuronal extracts were incubated with Sepharose beads conjugated with either wild-type D5 peptide (D5 wt) or D5 mutant 1 peptide with alterations in both FD and FV (D5 mt). A, Proteins eluted with EDTA from wild-type or mutant D5 peptide were separated on 8% SDS-PAGE gels under nonreducing conditions and transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody against α7 integrin chain or polyclonal antibody against β1 integrin chain. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots of proteins eluted with EDTA from D5 wt peptide revealed polypeptide bands with Mr of 120,000 and 116,000, respectively, for the O26 and β1 integrin antibodies. Blots of proteins eluted with EDTA from D5 mt peptide revealed no polypeptide bands. B, Silver-stained gels revealed overlapping polypeptide bands with Mr of 120,000 and 116,000 for proteins eluted with EDTA from D5 wt peptide but not D5 mt 1 peptide.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Incubation, Mutagenesis, SDS Page, Staining

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 1. Increased expression of b1,6GlcNAc residues on b4-integrin in human carcinoma cells. A, Glycosylation reactions catalyzed by glycosyltransferases, GnT-III and GnT-V. b1,6GlcNAc-branched N-glycans can be preferentially modified by polylactosamine N-glycan containing b-galactoside. Bisecting GlcNAc N-glycans inhibit b1,6GlcNAc- branching formation catalyzed by GnT- V. B, Immunohistochemical analysis of bisecting GlcNAc and b1,6GlcNAc expression in human cutaneous SCC using E4-PHA and L4-PHA lectins. C, Immunofluorescence microscopic analysis of b4-integrin (red) and b1,6GlcNAc (L4-PHA, green) in paraffin- embedded sections of human cutaneous SCC. Merged images are shown with nuclear Hoechst staining (blue). Normal human skin was stained as comparison. D, Western blot analysis of b4-integrin immunoprecipitates from normal human keratinocytes (NHK) and Ras/IkB-transformed NHK (transformed NHK) with L4-PHA and E4-PHA lectins, and anti–b4-integrin polyclonal antibody. Results of the densitometric analysis are shown as the integrated density of lectin-recognized b4 to total b4 bands, which was 1.0 for NHK cells.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Expressing, Glycoproteomics, Immunohistochemical staining, Staining, Comparison, Western Blot, Transformation Assay

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 2. Decreased b1,6GlcNAc residues in b4- integrin by GnT-III correlates with suppression of cancer cell migration and tumorigenesis. A, Cell surface expression of b4-integrin in MDA- MB435S cells expressing control lacZ, lacZ with GnT-III (lacZþGnT-III), b4, and b4 with GnT-III (b4þGnT-III). B, Lectin blot analysis of b4-integrin immunoprecipitates from MDA- MB435S cells expressing b4 and b4 with GnT-III using L4-PHA (b1,6GlcNAc) and E4-PHA (bisecting GlcNAc) lectins. Data are representative of three independent experiments. C, Effect of GnT-III overexpression on b4-integrin– mediated cell migration. Results are the mean SEM of three independent experiments conducted in triplicate. , P < 0.001. ns, not significant (two- way ANOVA, Bonferroni posttest). D, Tumor formation in nude mice injected subcutaneously with the indicated cells. Results are the mean SEM of 5 mice per group. , P < 0.05 (two-way ANOVA, Bonferroni posttest).

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Migration, Expressing, Control, Over Expression, Injection

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 3. N-Glycosylation of b4-integrin drives tumor cell proliferation, migration, and invasion through PI3K/Akt activation. A, Schematic diagram of the human b4-integrin cDNA constructs in this study. WT, wild-type b4-integrin; DN, N-glycosylation–defective b4-integrin mutant. The sites corresponding to the N-glycosylation sites on b4-integrin subunit (Asn327, Asn491, Asn579, Asn617, and Asn695) are shown by flags. Numbers indicate the number of amino acid residues. TM, transmembrane region. B, Lectin blot analysis of b4-integrin immunoprecipitates from MDA-MB435S cells expressing lacZ, WT, and DN with L4-PHA, E4-PHA, and SSA lectins. C, Cell adhesion and spreading of MDA-MB435S transfectants to laminin-332. Note that MDA-MB435S cells expressing no b4-integrin (lacZ) or DN showed decreased cell adhesion and spreading compared with the cells expressing WT. Cell spreading morphology was examined under a phase-contrast microscope after incubation for 20 minutes. D–F, Effect of the N-glycosylation defect in b4-integrin on cell proliferation (D), migration (E), and matrigel invasion (F). Note that cells expressing DN showed decreased cell proliferation, migration, and matrigel invasion, which were rescued by overexpression of constitutively active PI3K p110a subunit (DNþPI3K). In contrast, overexpression of the active PI3K subunit had no significant effect on WT-b4–dependent cell proliferation, migration, or matrigel invasion (WTþPI3K). Results are the mean SEM for three independent experiments conducted in triplicate. , P < 0.01; , P < 0.001; ns, not significant (one-way ANOVA, Bonferroni posttest) versus WT. G, Western blot analysisof cell lysates from MDA-MB435S transfectantswith phospho-Akt and total Akt antibody. Note that cells expressing lacZ or DN showed decreased PI3K/Akt pathway activation, which was rescued by overexpression of constitutively active p110a PI3K subunit (DNþPI3K). Results of the densitometric analysis are shown as the integrated density of the ratio of phosphorylated protein to total protein bands. All blots are representative of at least three independent experiments.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Glycoproteomics, Migration, Activation Assay, Construct, Mutagenesis, Expressing, Microscopy, Incubation, Over Expression, Western Blot

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 4. N-Glycosylation of b4-integrin functionally contributes to tumorigenesis in vivo. A, Tumor growth after subcutaneous injection of MDA-MB435S cells expressing the indicated proteins into mice (n ¼ 5 mice per group SEM). , P < 0.01; , P < 0.001 (one-way ANOVA, Bonferroni posttest). B, Hematoxylin/ eosin staining of the indicated tumors. C, Immunofluorescent analysis of Ki-67 (green) and b4-integrin (red) expression in the indicated tumors. Nuclei were stained with Hoechst 33342 (blue). D, Quantified percentage of Ki-67-positive cells to total cells in C. The bar graph represents the mean SEM for three independent experiments. , P < 0.001; ns, not significant (one-way ANOVA, Bonferroni posttest) versus WT.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Glycoproteomics, In Vivo, Injection, Expressing, Staining

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 5. Galectin-3 is a key modulator of b4-integrin function through N-glycan. A, Inhibitory effect of a functional blocking antibody against galectin-3 (Ab) or control IgG on b4-integrin clustering. Note that MDA-MB435S cells expressing WT showed decreased b4-integrin clustering by the treatment of a functional blocking antibody against galectin-3. B, Inhibitory effect of a functional blocking antibody against galectin-3 on N-glycosylation of b4-integrin–mediated cell migration. Note that MDA-MB435S cells expressing WT but not DN showed decreased cell migration by the treatment of a functional blocking antibody against galectin-3. C, MDA-MB435S cells expressing WT and A431 cells were incubated in serum-free medium in the presence of IgG or a functional blocking antibody against galectin-3 (Ab) for 20 minutes. Cell lysates were analyzed by Western blot with phospho-Akt and total Akt antibody. D, Effect of galectin-3 (gal3) knockdown on tumor growth in MDA-MB435S cells expressing WT. Results are the mean SEM of six miceper group. , P < 0.001 (unpairedStudent t test). E, Western blot analysisof b4-integrin immunoprecipitates from MDA-MB435S transfectants and A431 cells. F, MDA-MB435S cells expressing WT and A431 cells were serum-starved for 2 hours and were then incubated in serum-free medium in the presence of conditioned medium of MDA-MB435S cells and saccharides (0.2 mol/L) for 30 minutes. The b4-integrin immunoprecipitates from the cells were analyzed by Western blot. Note that the binding of galectin-3 to b4-integrin was inhibited by a competitive disaccharide, b-lactose, but not a noncompetitive disaccharide, sucrose. Results of the densitometric analysis are shown as the integrated density of the ratio of galectin-3 protein to b4-integrin protein bands. G, Effect of galectin-3 on N-glycans of b4-integrin–mediated cell adhesion. Note that galectin-3 enhanced cell adhesion of MDA-MB435S cells expressing WT but not either lacZ or DN to laminin-332. H, Effect of galectin-3 on N-glycosylation in b4-integrin–mediated cell migration. Note that MDA-MB435S cells expressing WT, but not either WT with GnT-III or DN, showed increased cell migration in the presence of galectin-3. Results are the mean SEM for two or three independent experiments conducted in triplicate. , P < 0.05; , P < 0.001; ns, not significant (one-way ANOVA, Bonferroni posttest).

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Glycoproteomics, Functional Assay, Blocking Assay, Control, Expressing, Migration, Incubation, Western Blot, Knockdown, Binding Assay

Journal: Molecular Cancer Research

Article Title: β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3–N-Glycan Complex

doi: 10.1158/1541-7786.mcr-17-0365

Figure Lengend Snippet: Figure 6. Hypothetical model of N- glycosylation–mediated b4- integrin–dependent tumor progression. Galectin-3 cross-links between b1,6GlcNAc-branched N-glycans, which in turn promotes b4-integrin clustering. The complex formation activates PI3K/Akt signaling, thereby promoting tumor progression by stimulating cell migration, invasion, proliferation, and tumorigenesis.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies specific for galectin-3 (M3/38; #sc-2393; Santa Cruz Biotechnology), a6-integrin (GoH3; #sc-19622; Santa Cruz Biotechnology), and b4-integrin (439-9B; #555719; BD Transduction Laboratories); rabbit polyclonal antibodies against human b4-integrin (H101; #sc-9090; Santa Cruz Biotechnology), phospho-Akt (pSer473; #9271; Cell Signaling Technology), and Akt (#9272; Cell Signaling Technology); and mouse monoclonal antibodies against Ki-67 (#610968; BD Transduction Laboratories), and b4-integrin (3E1; #MAB1964; Merck Millipore).

Techniques: Glycoproteomics, Migration

Journal: Carcinogenesis

Article Title: Synthetic D-amino acid peptide inhibits tumor cell motility on laminin-5

doi: 10.1093/carcin/bgl005

Figure Lengend Snippet: HYD1 inhibits laminin-5-dependent cellular invasion. (A) Concentration-dependent inhibition of PC3N cell invasion toward laminin-5. Cells were added into the upper chamber of laminin-5-coated transwells for 1 h prior to treatment with 75, 100, 250 or 500 (μg/ml) HYD1 or HYDS in serum-free media as described in Materials and methods. (B) Comparison of HYD1 activity to other known inhibitors of invasion. Cells were added as in (A) and followed by treatment with 250 μg/ml HYD1 or HYDS in serum-free media. The Rho-kinase inhibitor, Y-27632, was used at a concentration of 25 μM in serum-free media. Integrin function-blocking antibodies P1B5 (α3), GoH3 (α6) and AIIB2 (β1) were used at a concentration of 10 μg/ml and were incubated with cells for 15 min at room temperature prior to adding the cells into transwells. Error bars are standard deviation from the mean. (C) Laminin subchain composition of laminin-5 matrix material. The concentrated conditioned media from HaCaT cells were analyzed by 6% PAGE, transferred to PVDF membranes and probed for the α3 chain (BM165, mouse monoclonal antibody), the β3 chain (BM140, mouse monoclonal antibody) or the γ2 chain (GB3, goat monoclonal antibody).

Article Snippet: GoH3, a rat anti-α6 functional-blocking antibody was purchased from Serotech (Raleigh, NC).

Techniques: Concentration Assay, Inhibition, Comparison, Activity Assay, Blocking Assay, Incubation, Standard Deviation

Journal: Carcinogenesis

Article Title: Synthetic D-amino acid peptide inhibits tumor cell motility on laminin-5

doi: 10.1093/carcin/bgl005

Figure Lengend Snippet: HYD1 inhibits laminin-5-dependent cellular invasion. (A) Concentration-dependent inhibition of PC3N cell invasion toward laminin-5. Cells were added into the upper chamber of laminin-5-coated transwells for 1 h prior to treatment with 75, 100, 250 or 500 (μg/ml) HYD1 or HYDS in serum-free media as described in Materials and methods. (B) Comparison of HYD1 activity to other known inhibitors of invasion. Cells were added as in (A) and followed by treatment with 250 μg/ml HYD1 or HYDS in serum-free media. The Rho-kinase inhibitor, Y-27632, was used at a concentration of 25 μM in serum-free media. Integrin function-blocking antibodies P1B5 (α3), GoH3 (α6) and AIIB2 (β1) were used at a concentration of 10 μg/ml and were incubated with cells for 15 min at room temperature prior to adding the cells into transwells. Error bars are standard deviation from the mean. (C) Laminin subchain composition of laminin-5 matrix material. The concentrated conditioned media from HaCaT cells were analyzed by 6% PAGE, transferred to PVDF membranes and probed for the α3 chain (BM165, mouse monoclonal antibody), the β3 chain (BM140, mouse monoclonal antibody) or the γ2 chain (GB3, goat monoclonal antibody).

Article Snippet: Antibodies and reagents GoH3, a rat anti-α6 functional-blocking antibody was purchased from Serotech (Raleigh, NC).

Techniques: Concentration Assay, Inhibition, Comparison, Activity Assay, Blocking Assay, Incubation, Standard Deviation