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: Arthritis Research & Therapy

Article Title: Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

doi: 10.1186/ar2024

Figure Lengend Snippet: Over-release of prostaglandin E2 (PGE 2 ) in compressed costal cartilage explants is the result of mechanical stress. (a) Implication of the mechanoreceptor integrin α5β1 in PGE 2 over-release in compressed cartilage explants. Mouse costal cartilage explants treated with either the β1 non-blocking antibody VMA1997 or the α5β1 blocking antibody AB1950 at 2.5 μg/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not-compressed control (cont) value. Data are the mean ± SEM of 2 independent experiments with n = 2/group/experiments, analyzed in duplicate. ***p < 0.001 versus control NC, *p < 0.05 versus control C. (b) Increased PGE 2 release in compressed costal cartilage explants is not due to the cytokine IL-1. Mouse costal cartilage explants treated with the IL-1 receptor antagonist (IL1-Ra) at 100 ng/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not compressed control value. Data are the mean ± SEM of 2 independent experiment with n = 2/group/experiments, analyzed in duplicate. *p < 0.05 versus control NC.

Article Snippet: Anti-goat fibronectin receptor (integrin α5β1) blocking polyclonal antibody (AB1950) was purchased from Euromedex for Chemicon Inc. (Strasbourg, France) and rat anti-mouse β1 subunit of VLA1 integrins non-blocking monoclonal antibody (VMA 1997), was purchased from AbCys SA for Chemicon Inc. (Souffelweyersheim, France).

Techniques: Blocking Assay

Journal: JCI Insight

Article Title: Targetable purinergic receptors P2Y 12 and A2b antagonistically regulate bladder function

doi: 10.1172/jci.insight.122112

Figure Lengend Snippet: Deletion of P2Y12 and A2b result in enlarged (A) and reduced (B) bladder size, respectively. (C–E) H&E staining images from bladder sections of wild-type, P2Y12-KO, and A2b-KO mice, respectively. (F–H) Immunostaining of Ki67 from bladder sections of wild-type (n = 5), P2Y12-KO (n = 5), and A2b-KO (n = 5) mice, respectively. Positive nuclear staining per bladder section is quantitated in I. (J–L) Integrin β1 immunostaining of bladder sections of wild-type (n = 186 cells), P2Y12-KO (n = 225 cells), and A2b-KO (n = 185 cells) mice, respectively. (M–O) Enlarged images of the white boxes seen in J, K, and L, respectively. (P) Schematic diagram indicating where the bladder was sectioned for staining, and how only bladder smooth muscle (BSM) cells containing nuclei were measured for their cross-section area, which serves as an index of BSM cell size. (Q) BSM cell cross-sectional area was quantitated for each model. (R and S) Quantitative RT-PCR data for c-fos and c-jun mRNA expression in mouse bladder from wild-type (n = 3), P2Y12-KO (n = 3), and A2b-KO (n = 3) mice. (T) Western blot images of c-fos and c-jun protein expression in mouse bladder from wild-type (n = 6), P2Y12-KO (n = 6), and A2b-KO (n = 6) mice, which are quantitated by densitometry and normalized to β-actin in U and V. Data are shown as box and whiskers, whiskers are from minimum to maximum, Student’s t test is used to compare between wild-type and KO animals; *P < 0.05.

Article Snippet: Fixed tissue was cryoprotected, frozen, sectioned, and incubated with rabbit polyclonal anti-Ki67 antibody (catalog ab15580, abcam) and purified rat anti-mouse β1 integrin antibody (catalog 550531, BD Bioscience) (1:100 dilution) overnight at 4°C.

Techniques: Staining, Immunostaining, Quantitative RT-PCR, Expressing, Western Blot