Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Oligonucleotide primers for RT-PCR amplification of integrin subunits

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Amplification

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Epithelial-expressed β1-integrin regulates IEC-6 adhesion. A: schematic diagram of laminin and collagen integrin receptor heterodimer partners. Squares indicate β-subunits, and circles represent α-subunit binding partners. B: integrin transcript was detected in rat intestinal epithelial cell line IEC-6. C: integrin transcript expressed in human intestinal cell lines, Caco2-BBe, T84, and HT29. H2O (negative control), RNA (RT negative control), and RNA isolated from rat small intestine (ratSI) or normal human colon tissue (positive control) were included in the analysis. D: functional β1-integrin blocking antibody (solid bars) decreases IEC-6 cell adhesion to laminin and collagen IV compared with untreated (open bars) or isotype (shaded bar) controls. Data are means ± SE from 3 experiments. Asterisk denotes statistically significant difference from untreated cells (*P ≤ 0.05).

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Binding Assay, Negative Control, Isolation, Positive Control, Functional Assay, Blocking Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: CXCL12 stimulation increases active-β1-integrin. A: representative histogram showing CXCL12 stimulation (2.5 nM) (shaded region) for 1 h at 37° increased active-β1-integrin on the cell surface of Caco2-BBe cells compared with unstimulated cells (gray line). MnCl2 (1 mM) (gray dashed line) synthetically produced maximal β1-integrin activation. B: quantification of active β1-integrin on Caco2-BBe cells that were untreated (no stim, open bars) stimulated with 2.5 nM CXCL12 (solid) or incubated with synthetic controls MnCl2 (hatched bar) or EDTA (5 mM) (shaded bar). Data are expressed as the mean percentage of unstimulated mean fluorescence intensity ± SE of 5–6 experiments. Asterisk denotes statistically significant difference from untreated cells (*P ≤ 0.05, **P ≤ 0.01).

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Produced, Activation Assay, Incubation, Fluorescence

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: α3-Integrin depletion within enterocytes. A: IEC-6 cells were equally transduced using LL3.7 green fluorescence protein (GFP) vectors, which were empty (GFP-Mock), contained a scrambled sequence (GFP-Scrambled), or shRNA specific for rat α3-integrin (GFP-shα3 Integrin). WT, wild-type. B: representative immunoblot detecting α3 and other epithelial integrins within WT, transduction controls, and GFP-shα3 integrin IEC-6 cells. C: densitometric analysis showing significant depletion of α3-integrin in GFP-shα3 cells (solid bar) compared with WT (open bar) and transduction controls (shaded bars). Data expressed as relative α3-integrin level normalized to GAPDH loading control from 9 different analyses. Asterisk denotes statistically significant difference from WT cells (***P ≤ 0.001) measured by ANOVA. Scale bar = 200 μm.

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Fluorescence, Sequencing, shRNA, Western Blot, Transduction

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Depletion of α3-integrin decreased basal cell restitution through altered linear migration persistence. A: live cell imaging analysis reveals that the basal migration rate of GFP-shα3 integrin (■) knockdown IEC-6 cells is diminished compared with WT cells (○). Data are means ± SE distance from start from 4 individual experiments with 8–10 cell tracks measured per experiment. B: representative time-lapsed images from 0, 6, 12 and 18 h. C: representative migration track overlays of WT and GFP-shα3 integrin IEC-6 cell migration. D: cell trajectories for all cells measured within the 4 individual experiments (WT, n = 32; GFP-shα3 integrin, n = 40). E: persistence of cell migration for WT (open bars) and GFP-shα3 integrin (solid bars) IEC-6 cells. Data are the means ± SE of 4 individual experiments with 8–10 cell tracks measured per experiment. Scale bar = 125 μm. Asterisk denotes statistically significant difference from untreated cells (*P ≤ 0.05, **P ≤ 0.01).

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Migration, Live Cell Imaging

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Depletion of α3-integrin prevents inducible cell restitution. A: migration-inducing stimulants CXCL12 (2.5 nM) (solid bars) or transforming growth factor (TGF)-β1 (5 ng/ml) (hatched bars) were unable to increase migration of GFP-shα3 integrin-depleted IEC-6 cells. Control WT, GFP-Mock, and GFP-Scrambled control transduced cells had significant induction of migration by both CXCL12 and TGF-β1 compared with unstimulated (open bars) controls. Data are means ± SE of 9 individual experiments. B: representative photomicrographs of WT, GFP-Mock, GFP-Scrambled, and GFP-shα3 integrin IEC-6 cells stimulated with CXCL12, TGF-β1, or left untreated (no stim). Scale bar = 125 μm. C: representative immunoblots show depletion of TGF-β receptor 1 (TGFβR1), whereas CXCR4 levels were unaffected by α3-integrin depletion. D: densitometric quantification of TGFβR1 in WT (open bar), GFP-Mock, GFP-scrambled (shaded bars), and GFP-shα3 integrin (solid bar) IEC-6 cells confirmed significant depletion of TGFβR1 in GFP-shα3 integrin cells. Data are expressed as relative TGFβR1 levels normalized to the GAPDH loading control from 7 different immunoblot analyses. Asterisk denotes statistically significant difference from WT cells (*P ≤ 0.05). E: caspase 3/7 activity in WT, GFP-Mock, GFP-scrambled, and GFP-shα3 integrin IEC-6 cells was assessed using a luciferase-based assay. Gliotoxin (2 μg/ml) treatment was used to induce cell death, whereas zVAD (10 μg/ml), a pan-caspase inhibitor, confirmed that gliotoxin-induced cell death was the result of caspase activity. Values are means ± SE of 3 individual experiments.

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Migration, Western Blot, Activity Assay, Luciferase

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: α6-Integrin is required for CXCL12-stimulated migration. A: representative immunoblot analyses of WT, pLKO-scrambled and pLKO-shα6 integrin IEC-6 cells showed decreased α6, whereas level of other integrins was unaffected. B: quantification of α6 reduction in WT, pLKO-scrambled, pLKO-shα6 IEC-6 cells. Values are relative α6 expression normalized to GAPDH loading control compared with WT cells from 6 different immunoblot analyses. C: CXCL12 stimulation (2.5 nM) (solid bars) was unable to stimulate increased migration in α6-depleted IEC-6 cells, whereas 5 ng/ml TGF-β1 (hatched bars) stimulated significantly more migration than untreated cells (open bars). Inducible migration within WT and pLKO-scrambled control cells remained intact. Values are means ± SE of 6 individual experiments. Asterisk denotes statistically significant difference from WT or untreated cells (*P ≤ 0.05, **P ≤ 0.01). Scale bar = 125 μm.

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Migration, Western Blot, Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Functional blockade of α6-integrin function blocks CXCL12-stimulated migration of Caco2-BBe cells

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Functional Assay, Migration

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

doi: 10.1152/ajpgi.00208.2011

Figure Lengend Snippet: Depletion of laminin-specific α3 and α6 blocks CXCL12-stimulated cell spreading. A and C: CXCL12-stimulated (2.5 nM) (solid bar) cell spreading was attenuated in GFP-shα3 integrin and pLKO-shα6 integrin expressing IEC-6 cells, whereas EGF stimulation (50 ng/ml) (hatched bar) was still able to evoke increased cell spreading. B and D: representative images of GFP-shα3 integrin and pLKO-shα6 integrin IEC-6 cells spreading on laminin. E and F: cell spreading was little affected by lentiviral gene transduction, as CXCL12- and EGF-induced functions remained intact in GFP-scrambled and pLKO-scrambled IEC-6 cells. Values are means ± SE of 3–4 individual experiments. Asterisk denotes statistically significant difference from untreated cells (*P ≤ 0.05, **P ≤ 0.01). Scale bar = 50 μm.

Article Snippet: Purified hamster anti-rat β1-integrin and hamster IgM, as well as phycoerythrin-conjugated anti human active-β1-integrin (clone HUTS-21) ( 35 ) and mouse IgG2a isotype, were purchased from BD Pharmingen (San Diego, CA).

Techniques: Expressing, Transduction

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept

doi: 10.4137/CMAMD.S34424

Figure Lengend Snippet: Comparison of MAPK expression of bone marrow by abatacept.

Article Snippet: The experimental primary antibodies were described earlier, such as anti-TNF-α mouse monoclonal antibody (1:1,000 dilution; Biogenesis), antihuman IL-6 rabbit polyclonal antibody (1:500, Rockland), antihuman CD4, anti-human CD68 mouse monoclonal antibody (1:1,000; DAKO), antihuman OPN mouse monoclonal antibody (1:250; Novo-castra), antihuman OPG, rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman RANKL (FL-317) rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman CD29 (beta-1 integrin) mouse monoclonal antibody (1:350; Novocastra), antihuman phospho-p38 MAPK (Tyr180/Tyr182) mouse monoclonal antibody (1:500; Cell Signaling), antihuman phospho-p44/42 MAPK (Tyr202/Tyr204, ERK1/ERK2) mouse monoclonal antibody (1:500; Cell Signaling), and antihuman phosphor-JNK (1:500; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept

doi: 10.4137/CMAMD.S34424

Figure Lengend Snippet: Comparison of MAPK expression of cartilage by abatacept.

Article Snippet: The experimental primary antibodies were described earlier, such as anti-TNF-α mouse monoclonal antibody (1:1,000 dilution; Biogenesis), antihuman IL-6 rabbit polyclonal antibody (1:500, Rockland), antihuman CD4, anti-human CD68 mouse monoclonal antibody (1:1,000; DAKO), antihuman OPN mouse monoclonal antibody (1:250; Novo-castra), antihuman OPG, rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman RANKL (FL-317) rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman CD29 (beta-1 integrin) mouse monoclonal antibody (1:350; Novocastra), antihuman phospho-p38 MAPK (Tyr180/Tyr182) mouse monoclonal antibody (1:500; Cell Signaling), antihuman phospho-p44/42 MAPK (Tyr202/Tyr204, ERK1/ERK2) mouse monoclonal antibody (1:500; Cell Signaling), and antihuman phosphor-JNK (1:500; Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept

doi: 10.4137/CMAMD.S34424

Figure Lengend Snippet: Immunohistochemical comparison of the expression of MAPK in bone (magnification, 200×; black arrow shows strong positive). ( A ) and ( B ): CD29 (β-1 integrin); ( C ) and ( D ): JNK; ( E ) and ( F ): ERK; ( G ) and ( H ): P38 MAPK. ( A ), ( C ), ( E ), and ( G ): control group (MTX); ( B ), ( D ), ( F ), and ( H ): abatacept group.

Article Snippet: The experimental primary antibodies were described earlier, such as anti-TNF-α mouse monoclonal antibody (1:1,000 dilution; Biogenesis), antihuman IL-6 rabbit polyclonal antibody (1:500, Rockland), antihuman CD4, anti-human CD68 mouse monoclonal antibody (1:1,000; DAKO), antihuman OPN mouse monoclonal antibody (1:250; Novo-castra), antihuman OPG, rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman RANKL (FL-317) rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman CD29 (beta-1 integrin) mouse monoclonal antibody (1:350; Novocastra), antihuman phospho-p38 MAPK (Tyr180/Tyr182) mouse monoclonal antibody (1:500; Cell Signaling), antihuman phospho-p44/42 MAPK (Tyr202/Tyr204, ERK1/ERK2) mouse monoclonal antibody (1:500; Cell Signaling), and antihuman phosphor-JNK (1:500; Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Expressing

Journal: Clinical Medicine Insights. Arthritis and Musculoskeletal Disorders

Article Title: Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept

doi: 10.4137/CMAMD.S34424

Figure Lengend Snippet: Immunohistochemical comparison of the expression of MAPK in cartilage (magnification, 200×; black arrow shows strong positive). ( A ) and ( B ): CD29 (β-1 integrin); ( C ) and ( D ): JNK; ( E ) and ( F ): ERK; ( G ) and ( H ): P38 MAPK. ( A ), ( C ), ( E ), and ( G ): control group (MTX); ( B ), ( D ), ( F ), and ( H ): abatacept group.

Article Snippet: The experimental primary antibodies were described earlier, such as anti-TNF-α mouse monoclonal antibody (1:1,000 dilution; Biogenesis), antihuman IL-6 rabbit polyclonal antibody (1:500, Rockland), antihuman CD4, anti-human CD68 mouse monoclonal antibody (1:1,000; DAKO), antihuman OPN mouse monoclonal antibody (1:250; Novo-castra), antihuman OPG, rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman RANKL (FL-317) rabbit polyclonal antibody (1:200; Santa Cruz Biotechnology), antihuman CD29 (beta-1 integrin) mouse monoclonal antibody (1:350; Novocastra), antihuman phospho-p38 MAPK (Tyr180/Tyr182) mouse monoclonal antibody (1:500; Cell Signaling), antihuman phospho-p44/42 MAPK (Tyr202/Tyr204, ERK1/ERK2) mouse monoclonal antibody (1:500; Cell Signaling), and antihuman phosphor-JNK (1:500; Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG protein is associated with β1 integrin and activates β1 integrin. a - b . Coimmunoprecipitation. Cell membrane proteins of HCT116 cells were collected and subjected to immunoprecipitated (IP) using anti-SNCG ( a ), anti-SNCG or anti-β1 integrin antibody ( b ). The IP proteins or total cell lysates were analyzed by Western blot. Normal IgG served as the negative control. c . Far-Western blot analysis. HCT116 cells were transfected with control siRNA (lane 1-2), and specific siRNA-β1-2 (lanes 3-4) for 48 h. Cells were treated without (lane 1, 3) or with 1 μmol/L rhSNCG (lane 2, 4). Cell lysates were subjected to SDS-PAGE and transferred to NC membrane. β1 integrin (prey protein) on the membrane is detected with SNCG (bait protein). More SNCG was associated with membrane β1 integrin in SNCG-treated cells than that in the control cells (lane 1, 2). Correspondingly, less SNCG were detected in β1 integrin knock-down cells than that in control cells (lane 2, 4). d - e , Effect of concentration and time treatment of SNCG on activated β1 integrin. HCT116 cells were stimulated with GST or GST-SNCG at various concentrations for 60 min ( d ) or at fixed concentration (1 μmol/L) for various times ( e ). Cell lysates were analyzed with the HUTS-21 mAb recognizing the activated form of β1 integrin. f , GST-SNCG treatment (1 μmol/L) upregulated activated β1 integrin subunit in HCT116 and SW480 cells. g , Colocalization of SNCG with F-actin. HCT116 cells grown on coverslips were transiently transfected with control siRNA or β1-specific siRNA-2. After 72 h, cells were treated with GST or GST-SNCG (1 μmol/L) for 60 min. Cells were fixed and stained with anti-SNCG (red) and FITC-Phalloidin (green). Colocalization of SNCG and F-actin was shown in yellow. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Far Western Blot, Transfection, SDS Page, Concentration Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Integrin β1 is required for enhancement of SNCG on tumor cell migration and invasion. a - b , The functional blocking antibody for β1 integrin subunit (5, 10, 20 μg/mL) was added in the upper compartment of migration or invasion chambers stimulated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( a ) or 48 h for invasion ( b ). c - d , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. then cells were treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( c ) or 48 h for invasion ( d ). e - f , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min and then treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( e ) or 48 h for invasion ( f ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. g , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot. h , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Functional Assay, Blocking Assay, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: FAK is essential for SNCG-enhanced tumor cell migration and invasion. a - b , HCT116 cells were transfected with control siRNA and FAK-specific siRNA-2, -3 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for migration ( a ) or invasion ( b ). c - d , HCT116 cells were treated with PBS, 50 μmol/L FAK inhibitor 14 for 30 min and then treated with or without GST-SNCG (1 μmol/L) for migration ( c ) or invasion ( d ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. e - f , HCT116 cells were transfected with control siRNA (lane 1-2), and FAK-specific siRNA-2 (lane 3-4) and -3 (lane 5-6) for 72 h. cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed for activated and total β1 integrin ( e ) or activated and total FAK ( f )

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG is an indicator of adverse prognosis and positively correlates with activated β1 integrin, p-FAK (Y 397 ) in CRC tissues. a - b , Kaplan-Meier estimation of disease-free survival (DFS) for stage I-II ( a ) and III-IV ( b ) colorectal adenocarcinoma patients according to SNCG levels. c , Correlations of SNCG levels in CRC tissues with post-operative recurrence and status. d , Representative blots from three independent experiments were presented. Protein levels of SNCG, activated β1 integrin, and p-FAK (Y 397 ) in clinical colon cancer tissue samples were evaluated by Western blot analysis. In order to increase the reproducibility, HCT116 cell lysates were used in each blot as the internal control ( c ) to minimize the effect of band intensity variation. GAPDH was used as the loading control. e - g , Correlation between the relative protein levels of activated β1 integrin levels and p-FAK (Y 397 ) ( e ), SNCG and active β1 integrin ( f ), and SNCG and p-FAK (Y 397 ) ( g ) were plotted as a scatter plots

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Exogenously added SNCG remodels the microenvironment of tumor cells and increases MMP-2 activity by β1 integrin. a , Antibody array screening of CM from GST and GST-SNCG-treated HCT116 cells. b , CM (left panel) and whole cell lysates (right panel) from HCT116 and SW480 cells treated with or without GST-SNCG (1 μmol/L) were subjected to Western blot analysis. Representative blots from three independent experiments were presented. c - d , HCT116 cells were treated with diluent, 50 μmol/L MMP-2 inhibitor for 40 min, then 1 μmol/L GST or GST-SNCG was added in the cell medium for migration ( c ) or invasion ( d ) assay. Migrated or invaded cells were quantitated after 24 h or 48 h, respectively. Error bars, SE of three determinations. e - f , Western blot and gelatin zymography analysis. CM from HCT116 cells treated with GST or GST-SNCG (1 μmol/L) in β1 integrin knock-down ( e ) or RGD-treated cells ( f ) was analyzed by gelatin zymography with FBS as the positive control, and secreted protein levels were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Activity Assay, Ab Array, Western Blot, Migration, Zymography, Positive Control

Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A Schema, (1) CRISPR screen. 26 ARF6‐proximal proteins from TurboID studies were investigated for their contribution to ARF6‐mediated invasion of ID8 Trp53 −/− ; Pten −/− spheroids. (2) For each interactor, 5 sgRNAs were cloned into lentiviral CRISPR vectors. (3) A pooled approach was used, generating a KO cell line with all 5 sgRNAs (4) Live imaging performed. (5) Phenotype of each KO compared with nontargeting sgRNA. B Frequency of Spherical and Hyper‐protrusive phenotypes upon pooled gRNA CRISPR of indicated targets (sorted based on hierarchical clustering) in ID8 Trp53 −/− ; Pten −/− clone 1.15 cells, performed in four parts (Iterations indicated). Heatmap (grayscale)—phenotype proportion ( z ‐score) in control (sgNT). Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, nonsignificant). N = 3–4 independent experiments, 3–6 technical replicates/experiment. Total spheroid number per condition, Table . C Western blot, β1‐integrin (ITGB1), pS473‐AKT, AKT, ARF6 from deconvolved ITGB1 sgRNA‐expressing cells. VCL, loading control for ITGB1, sample integrity control for other blots. Representative blots of n = 3 independent lysate preparations. D Quantitation of (C). Data, mean ± SD for pS473‐AKT:total AKT band intensity ratio, total AKT or ARF6 intensity, normalised to control (sgNT ID8 Trp53 −/− ; Pten −/− clone 1.15) cells. P ‐values, unpaired, two‐tailed t ‐test. E, F Frequency of Spherical and Hyper‐protrusive phenotypes in ID8 Trp53 −/− ; Pten −/− 1.15spheroids upon CRISPR‐mediated KO of (E) Itgβ1 or (F) Agap1 , 6 h time intervals over 72 h. Heatmap (grayscale)—phenotype proportion ( z ‐score) in control (sgNT). Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, non‐significant). N = 3 independent experiments, 1–5 technical replicates/experiment. Total spheroid number per condition, Table . G Representative phase contrast images of spheroids described in (E, F). Outlines pseudocoloured for classification (Spherical, green; Hyper‐protrusive, blue) at indicated timepoints. Magnified individual spheroids from boxed regions. Arrowheads, protrusions into ECM. Scale bars, 400 or 17 μm, as indicated. H Representative confocal images of Trp53 −/− and Trp53 −/− ; Pten −/− clone 1.15 spheroids expressing sgNT, sg Agap1 (sg3) or sg Itgb1 (sg4), stained for collagen IV (grayscale) and F‐Actin (magenta). Boxed areas, basement membrane region in higher magnification. Arrowheads, Collagen IV labelling that is: well‐defined, green; fragmented, yellow; absent, navy. Scale bar, 53 μm. I Quantitation of (H). Collagen IV basement membrane staining as Defined, Fragmented, or Absent in spheroids set up across n = 3 independent experiments, 1 technical replicate/experiment, 5–9 fields imaged per technical replicate, 365 spheroids scored in total. Data, mean ± SD of % of spheroids in each phenotype for independent experiments, with circles representing technical replicates. Unpaired t ‐test, P ‐values annotated. Source data are available online for this figure.

Article Snippet: Antibodies used were: anti‐2A peptide (Merck, MABS2005, 3H4), anti‐AKT pan (CST, 2920, 40D4), anti‐ARF5 (Novus Biologicals, H0000281‐M01, IB4), anti‐ARF6 (Merck, A5230), anti‐GAPDH (CST 2118, 14C10, 1:5,000), anti‐α5 integrin (Abcam, ab150361), anti‐β1 integrin (Merck, clone MB1.2), anti‐AKT phospho S473 (CST, 4060, D9E), anti‐S6RP phospo S235/236 (CST, 2217, 5G10), anti‐S6RP (CST, 2217, 5G10), PTEN (CST, 9552), anti‐RFP (used to detect BFP; Thermo Fisher Scientific, R10367), anti‐Streptavidin‐Horseradish Peroxidase (HRP) Conjugated (Thermo Fisher Scientific, SA10001), anti‐TP53 (Abcam, ab26, diluted in 5% milk in TBS‐T), anti‐V5 Tag (ABM, G189), anti‐Vinculin (Merck, V9131, 1:2,000).

Techniques: CRISPR, Clone Assay, Imaging, Western Blot, Expressing, Quantitation Assay, Two Tailed Test, Staining, Membrane

Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A, B Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for α5‐integrin or β1‐integrin (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n = 3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F‐actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. C, D Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n = 5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F‐actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. E–H Representative capture ELISA graphs (E, G) and associated quantitation (F, H) for recycling of internalised cargoes between Trp53 −/− versus Trp53 −/− ; Pten −/− cells or Trp53 −/− ; Pten −/− cells expressing sh Scramble versus sh Arf6 for active β1‐integrin. Graphs shown are representative of n = 2 (E) or n = 3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1–3 technical replicates/experiment/timepoint (small circles), two‐tailed t ‐test, P ‐values are annotated. I–K Overall survival (% patients, months; TCGA OV data set) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1 + Q2, Q3, low PTEN, green Q4, high PTEN). Median survival, sample size ( n ) and P ‐value, Log‐rank test (Mantel‐Cox) annotated. L Differential abundance ( x , Log Ratio between conditions; y , Log 10 q ‐values) of proteins in PIP 3 ‐responsive module (ARF6 HI ‐AGAP1 HI ‐CYTH2 2G ) versus PI(4,5)P 2 ‐responsive ARF module (ARF6 HI ‐AGAP1 HI ‐CYTH2 3G ) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (−Log 10 q ‐value > 1.3). M Schema, molecular model for ARF GTPase regulation of integrin‐dependent invasion. Source data are available online for this figure.

Article Snippet: Antibodies used were: anti‐2A peptide (Merck, MABS2005, 3H4), anti‐AKT pan (CST, 2920, 40D4), anti‐ARF5 (Novus Biologicals, H0000281‐M01, IB4), anti‐ARF6 (Merck, A5230), anti‐GAPDH (CST 2118, 14C10, 1:5,000), anti‐α5 integrin (Abcam, ab150361), anti‐β1 integrin (Merck, clone MB1.2), anti‐AKT phospho S473 (CST, 4060, D9E), anti‐S6RP phospo S235/236 (CST, 2217, 5G10), anti‐S6RP (CST, 2217, 5G10), PTEN (CST, 9552), anti‐RFP (used to detect BFP; Thermo Fisher Scientific, R10367), anti‐Streptavidin‐Horseradish Peroxidase (HRP) Conjugated (Thermo Fisher Scientific, SA10001), anti‐TP53 (Abcam, ab26, diluted in 5% milk in TBS‐T), anti‐V5 Tag (ABM, G189), anti‐Vinculin (Merck, V9131, 1:2,000).

Techniques: Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Expressing, Two Tailed Test, Protein Array

Journal: Molecular cancer research : MCR

Article Title: Heparanase – induced GEF-H1 signaling regulates the cytoskeletal dynamics of brain metastatic breast cancer cells

doi: 10.1158/1541-7786.MCR-11-0534

Figure Lengend Snippet: BMBC demonstrate differential GEF-H1 and PKCα protein expression and HPSE treatment modulated SDC4 CT association. A. GEF-H1 and PKCa are SDC CT associated proteins in HPSE-treated BMBC cells. Whole cell lysates from BMBC cells treated with L- or A-HPSE were immunoblotted for GEF-H1, PKCa, SDC4 or β1Integrin to determine protein expression levels in different BMBC cell lines. Blots were probed for β-actin as a loading control. Data presented are representative of four reproducible experiments. B. The production of GST fusion proteins. Affinity glutathione-S-transferase pulldowns for SDC1 and SDC4 were performed as previously described (11). Figure indicates eluted lysates (1 mg/lane) run on a 15% SDS-PAGE gel under reducing conditions. C. GST pulldown of GEF-H1 by SDC1/4CT. D. GST pulldowns of PKCa by SDC1/4CT. BMBC cells were treated with or without latent or active (L- or A-HPSE) (100ng/ml for 1 hr. at 37°C), then whole cell lysates were generated, passed over GST-SDC1/4 CT fusion protein affinity columns. The same lysates were loaded onto GST protein affinity columns followed by immunoblotting for GEF-H1 or PKCa as controls. Refer to the “Materials and Methods” section for additional details.

Article Snippet: The antibodies and dilutions used in experiments were: SDC1 (1:1,000, clone B-A38), Grb2 (1:1,000, 3972), purchased from Cell Sciences (Canton, MA), SDC4 (1:1,000, ab24511) was from Abcam (Cambridge, MA), full-length PKCα (1:1,000, #2506), GEF-H1 (1:1,000, #4076) and GAPDH (1:1,000, 14C10) were from Cell Signaling (Danvers, MA), β1Integrin (1:1,000, clone 656) was from BD Biosciences (Sparks, MD), Rac1 (1:1,000, clone 23A8) and RhoA (1:500, clone 55) were from Millipore (Danvers, MA), Tiam1 (1:1,000, sc872) and β-actin (1:1,000, sc-69879) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, SDS Page, Generated, Western Blot