Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: To pharmacologically block Rho/ROCK pathway and β1-integrin, hPAECs were pretreated with the ROCK inhibitor Y-27632 (#10005583, Cayman Chemical Company, Michigan) and an anti-β1-integrin subunit antibody (#MAB17781, clone P5D2, R&D Systems, Minnesota).

Techniques: Activation Assay, Expressing, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: Aberrant Activation of Focal Adhesion Proteins Mediates Fibrillar Amyloid β-Induced Neuronal Dystrophy

doi: 10.1523/JNEUROSCI.23-02-00493.2003

Figure Lengend Snippet: Fibrillar Aβ induces FA-like structures in dystrophic neurons. Cortical neurons were treated with fibrillar Aβ at day 5, fixed at day 7, and stained with phalloidin-Texas Red, anti-Aβ, anti-paxillin (pax), and anti-integrin (int) antibodies. FAs were identified by colocalization of paxillin or integrin clusters with microfilaments.A–D, FA are absent in neurons grown on poly-l-lysine (control, PLL). Neuronal processes exhibit homogeneous distribution of paxillin and integrin, whereas microfilaments (MF, red) are primarily localized in growth cones (A, C, arrows). E, F, Neurons grown on laminin (control, Lam) exhibit paxillin and integrin clusters (arrows), associated with microfilaments periodically localized along the processes.G–J, Aβ-treated neurons on poly-l-lysine [Aβ (PLL)] develop FA-like structures proximal to Aβ fibrils (blue) that include clusters of paxillin and integrin (arrows). Microfilaments (MF, red) protruding from FA-like structures are evident in G and H (arrowheads). In some cases, growth cone filopodia appear to reverse orientation and extend toward Aβ deposits (I, J, arrowheads).K–N, Phospho-Tyr immunoreactivity (Tyr-P, green) colocalizes with paxillin (blue) and microfilaments (MF, red) in dystrophic processes (arrows). Scale bar: (in F)A–N, 5 μm. O, Quantification of integrin and paxillin clustering. Integrin receptor clustering increased 2.0 ± 0.2-fold on a laminin substrate and 1.8 ± 0.2-fold in neurons grown on poly-l-lysine after Aβ treatment. Paxillin clustering increased 3.0 ± 0.3-fold on a laminin substrate and 2.5 ± 0.3-fold in neurons grown on poly-l-lysine after Aβ treatment. Values are mean ± SEM; n = 3 independent experiments; >100 FA contacts were scored per condition; *p < 0.05 relative to control by ANOVA followed by the Student–Newman–Keulspost hoc test.

Article Snippet: The following primary antibodies were used: anti-α5β1-integrin receptor (1:100), rabbit anti-β1-integrin subunit (1:40; Chemicon, Temecula, CA), PHF-1, which recognizes phosphorylated tau at Ser-396 and Ser-404 (1:500; Greenberg et al., 1992 ), mouse anti-Aβ 6E10 (1:500; Senetek, Napa, CA), rabbit anti-paxillin phosphorylated at residue 31 (1:100; Biosource), and rabbit anti-FAK phosphorylated at Tyr-397 (1:100; Biosource).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Aberrant Activation of Focal Adhesion Proteins Mediates Fibrillar Amyloid β-Induced Neuronal Dystrophy

doi: 10.1523/JNEUROSCI.23-02-00493.2003

Figure Lengend Snippet: Expression of FA proteins in Alzheimer's brain. Paraffin-embedded brain sections of AD and age-matched control specimens were silver-stained or immunolabeled. A, In AD brains, silver staining revealed dystrophic neurites (black) in senile plaques, surrounding the core of Aβ (pale yellow). B, Immunofluorescence shows the Aβ core of the plaque (green) surrounded by dystrophic neurites immunostained with antibody PHF-1, which recognizes hyperphosphorylated tau (blue). C, Integrin receptor immunoreactivity in a senile plaque (green) surrounding the Aβ core (blue). D, Hyperphosphorylated tau (blue) and integrin receptors (green) colocalize in dystrophic neurites and cell bodies in a senile plaque (arrows).E, Phosphorylated paxillin (pax-P,green) in dystrophic neurites and cell bodies in a senile plaque. The plaque core is stained with anti-Aβ antibody (Aβ, blue). F, Dystrophic neurites in a senile plaque immunostained with anti-phosphorylated FAK antibody (FAK-P, green) surrounding the Aβ core (blue). Adjacent brain sections silver-stained (G) and immunostained with anti-integrin antibody (int,H) show similar plaque density.Arrows denote individual plaques. I, Quantification of integrin- and hyperphosphorylated tau-positive plaques shows that 84 ± 6% of silver-stained plaques were positive for integrin immunoreactivity, and 79 ± 14% were positive for hyperphosphorylated tau. Ten to 20 microscopic fields were analyzed in adjacent sections of four AD brain cases. At least 50 plaques were scored per silver-stained section. Scale bars:A–F, 50 μm; G, H, 250 μm.

Article Snippet: The following primary antibodies were used: anti-α5β1-integrin receptor (1:100), rabbit anti-β1-integrin subunit (1:40; Chemicon, Temecula, CA), PHF-1, which recognizes phosphorylated tau at Ser-396 and Ser-404 (1:500; Greenberg et al., 1992 ), mouse anti-Aβ 6E10 (1:500; Senetek, Napa, CA), rabbit anti-paxillin phosphorylated at residue 31 (1:100; Biosource), and rabbit anti-FAK phosphorylated at Tyr-397 (1:100; Biosource).

Techniques: Expressing, Staining, Immunolabeling, Silver Staining, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Aberrant Activation of Focal Adhesion Proteins Mediates Fibrillar Amyloid β-Induced Neuronal Dystrophy

doi: 10.1523/JNEUROSCI.23-02-00493.2003

Figure Lengend Snippet: Model of the FA pathways involved in Aβ-induced neuronal dystrophy. Fibrillar Aβ binds to and induces the clustering of the integrin receptors, leading to the activation of paxillin and FAK and their translocation to the nascent FA complex. Paxillin binds to vinculin, which promotes microfilament stabilization at the FA site. PTP-PEST binds to paxillin, leading to dephosphorylation of several FA proteins, which prevents the stabilization of the FA contact, allowing the neuron to continuously respond to fibrillar Aβ stimuli. Alternatively, APP binds to Aβ fibrils, bringing them in contact with integrins, activating FA signaling through FE65, which binds to the C terminus of APP and associates with c-abl, which in turn binds and phosphorylates paxillin, or both. A pathway involving fyn, which is downstream of PTP-PEST, promotes GSK3β activity, whereas the interaction of cbl with c-abl increases CDK5 activity (Zukerberg et al., 2000). Both CDK5 and GSK3β hyperphosphorylate tau, leading to microtubular destabilization and neuronal dystrophy. An alternative pathway involving FAK activity leads to neuronal cell death but not neuronal dystrophy. Paxillin contains four SH2-binding domains (red), five Leu-rich LD domains (light blue), one Pro-rich SH3-binding domain (green) and four LIM domains (purple). MF, microfilaments;hyperphosp., hyperphosphorylated; P, phosphate groups.

Article Snippet: The following primary antibodies were used: anti-α5β1-integrin receptor (1:100), rabbit anti-β1-integrin subunit (1:40; Chemicon, Temecula, CA), PHF-1, which recognizes phosphorylated tau at Ser-396 and Ser-404 (1:500; Greenberg et al., 1992 ), mouse anti-Aβ 6E10 (1:500; Senetek, Napa, CA), rabbit anti-paxillin phosphorylated at residue 31 (1:100; Biosource), and rabbit anti-FAK phosphorylated at Tyr-397 (1:100; Biosource).

Techniques: Activation Assay, Translocation Assay, De-Phosphorylation Assay, Activity Assay, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Aberrant Activation of Focal Adhesion Proteins Mediates Fibrillar Amyloid β-Induced Neuronal Dystrophy

doi: 10.1523/JNEUROSCI.23-02-00493.2003

Figure Lengend Snippet: PTP-PEST binding to paxillin is required for Aβ-induced neuronal dystrophy. A, Aβ-induced neuronal dystrophy was assessed in neurons transfected with paxillin constructs bearing Cys-to-Ala point mutations, which disrupt the LIM domain tertiary structure, with FRNK, an FAK dominant negative construct, or with PESTdl, a PTP-PEST deletion construct.B, Mutations C470A, C467/470A, and C523A in paxillin, which prevent PTP-PEST binding and integrin association, significantly reduce Aβ-induced neuronal dystrophy (C470A, 62.6 ± 1.3%; C467/C470A, 21.2 ± 4.4%;C523A, 39.9 ± 18.5%). The mutationC467A, which also prevents PTP-PEST binding and integrin association, has no effect on Aβ-induced neuronal dystrophy (112.2 ± 36.1). Expression of PESTdl, which prevents PTP-PEST binding to paxillin, completely prevents neuronal dystrophy. Expression of FRNK, which contains the FAK focal adhesion targeting domain but lacks the kinase domain, has no effect on Aβ-induced neuronal dystrophy. The number of dystrophic neurons was quantified 24 hr after transfection and expressed as a percentage of the number of Aβ-induced dystrophic neurons expressing GFP (100%). Values are mean ± SEM; n = 3–7 individual experiments; 250 neurons were scored per condition in each experiment; *p < 0.05; **p < 0.01 relative to control (GFP) by ANOVA followed by the Student–Newman–Keuls post hoc test.

Article Snippet: The following primary antibodies were used: anti-α5β1-integrin receptor (1:100), rabbit anti-β1-integrin subunit (1:40; Chemicon, Temecula, CA), PHF-1, which recognizes phosphorylated tau at Ser-396 and Ser-404 (1:500; Greenberg et al., 1992 ), mouse anti-Aβ 6E10 (1:500; Senetek, Napa, CA), rabbit anti-paxillin phosphorylated at residue 31 (1:100; Biosource), and rabbit anti-FAK phosphorylated at Tyr-397 (1:100; Biosource).

Techniques: Binding Assay, Transfection, Construct, Dominant Negative Mutation, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Galectin-1 from cancer-associated fibroblasts induces epithelial–mesenchymal transition through β1 integrin-mediated upregulation of Gli1 in gastric cancer

doi: 10.1186/s13046-016-0449-1

Figure Lengend Snippet: β1 integrin is necessary for Gal-1-induced effects in GC cell migration, invasion, and EMT. a The silencing of β1-integrin expression by siRNA at 48 h was confirmed using western blotting ( left ) and qRT-PCR ( right ). * P < 0.05 when compared with the si-Control. b The expression of EMT-related molecules (E-cadherin, N-cadherin, vimentin, and snail), invasion-related molecules (MMP-9), key molecules in the Hh pathway (SHH, SMO, and Gli1) and β1 integrin were analyzed by western blotting ( left ). The mRNA expression levels were determined by qRT-PCR ( right ). * P < 0.05. The results are presented as the mean ± SD of three independent experiments. c MGC-803 cells were labelled with fluorescein-conjugated Gli1 specific antibody (green) following treatment with CM from Over (magnification × 200). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). d The invasion-promoting effects of Gal-1 were abolished by Gli1 knockdown. The number of invading cells was quantified under a light microscope by counting six random fields at a magnification of 200×. * P < 0.05. The data represent the results of three independent experiments

Article Snippet: Anti-galectin-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β1-integrin subunit antibody (Cell Signaling Technology, Danvers, MA, USA), anti-SMO antibody (Abcam, Cambridge, UK), anti-Gli1 antibody (Abcam), anti-SHH antibody (Abcam), anti-E-Cadherin antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Vimentin (Cell Signaling Technology), anti-Snail (Cell Signaling Technology), anti-MMP9 (Cell Signaling Technology), anti-β-actin antibody (Beyotime, Jiangsu, China), HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) were used in this study.

Techniques: Migration, Expressing, Western Blot, Quantitative RT-PCR, Control, Staining, Knockdown, Light Microscopy

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: Pull-down proteins were blotted with an anti-β1-integrin subunit antibody (#MAB1997, clone MB1.2, Merck-Millipore, Massachusetts).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: Subsequently, cells were divided into two equal parts and stained for 30 min with antibodies against total anti-β1-integrin subunit (10 μg/mL, #MAB1997, clone MB1.2, Merck-Millipore, Massachusetts) and against active anti-β1-integrin subunit (1:25, #MAB2247, clone 12G10, Merck-Millipore, Massachusetts).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation