Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Targeting apical integrins with AIIB2 nanowires produces a ruffled ZO-1 morphology. (A) Schematic of the general reduction and conjugation reactions that generate the antibody-decorated nanowires used in these experiments. (B) Representative immunofluorescence image of bare nanowires (labeled with Nile Red; red) on Caco-2 cells labeled with anti-ZO-1 antibodies (cyan). Scale bar: 10 μm. Image representative of five experiments. (C) Representative immunofluorescence images of Caco-2 cells labeled with anti-ZO-1 antibodies either untreated or 2 h after treatment with bare nanowires, IgG control nanowires, free reduced antibody (9EG7 or AIIB2), or anti-integrin nanowires (9EG7 or AIIB2). Scale bar: 30 μm. (D) Changes in ZO-1 morphology are quantified by measuring the ratio of the traced actual length between tricellular junctions (trace A) and the linear distance between those same junctions (trace B). (E,F) Quantification of junction length ratios (E) and percent of cells in a field of view with one or more ruffled junction (F) for each treatment displayed as mean±s.d. ( n =3 fields of view from three slides per condition; 25 measurements per field of view). Treatment key on left (Ab, antibody). Significance determined by one-way ANOVA with Fisher's LSD test. (E) # P =0.66 (1 versus 4); ** P =0.003 (2 versus 4); * P =0.028 (3 versus 4); * P =0.025 (1 versus 5); *** P =0.0008 (2 versus 5); ** P =0.009 (3 versus 5); ** P =0.0015 (4 versus 6); *** P =0.0004 (5 versus 6); **** P <0.0001 (7 versus all); ns, not significant. (F) **** P <0.0001 (1 versus 4); ** P =0.016 (2 versus 4); *** P =0.002 (3 versus 4); **** P <0.0001 (1 versus 5); *** P =0.0008 (2 versus 5); **** P <0.0001 (3 versus 5); **** P <0.0001 (4 versus 6, 5 versus 6, 7 versus all); ns, not significant.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Conjugation Assay, Immunofluorescence, Labeling, Control

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Targeting apical integrins with blocking nanowires produces a ruffled ZO-1 morphology. (A) The 9EG7 activating mAb (green) binds to the EGF domain locking an extended conformation. (B) The 12G10 activating mAb (green) binds to the βA domain locking an extended conformation. (C) The AIIB2 blocking mAb (blue) binds to the ligand binding site of the βA domain, irrespective of conformation. (D) The mAb13 blocking mAb (blue) binds to the βA domain locking a bent configuration. Ab, antibody; β1, integrin β1. (E) Representative immunofluorescence images of Caco-2 cells labeled with anti-ZO-1 antibodies either untreated or 2 h after treatment with the indicated anti-integrin nanowires. Scale bar: 10 μm. (F,G) Quantification of junction length ratios (F) and percent of cells in a field of view with one or more ruffled junctions (G) for each treatment displayed as mean±s.d. ( n =4 or 5 fields of view from two slides per condition; 15 measurements per field). Treatment key on right. Significance determined by one-way ANOVA with Fisher's LSD test. (F) * P =0.015 (untreated versus 9EG7); * P =0.016 (untreated versus 12G10); **** P <0.0001; ns, not significant. (G) * P =0.031 (untreated versus 9EG7); # P =0.066 (untreated versus 12G10); *** P =0.001 (untreated versus mAb13); **** P <0.0001; ns, not significant.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Blocking Assay, Ligand Binding Assay, Immunofluorescence, Labeling

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Time course and reversibility of the effects of AIIB2 nanowires on tight junctions. (A) Representative immunofluorescence images of Caco-2 cells labeled with anti-ZO-1 antibodies and fixed at multiple time points after treatment with AIIB2 nanowires. Scale bars: 10 μm. (B) Quantitation of junction length ratios for both treatments at each time point showed that significant ZO-1 ruffling occurred 1 h after application of nanowires. Data displayed as mean junction length ratio+s.d. ( n =50 measurements per treatment per time point). Significance was determined by two-way ANOVA with Bonferroni's correction for multiple comparisons. **** P <0.0001. (C) Schematic of the GSH competition assay where cells were incubated with AIIB2 nanowires for 2 h, followed by addition of 1 mM GSH, which cleaves antibody conjugates off the wires over an 8 h incubation period. β1, integrin β1. (D) Quantitation of the number of cell-associated nanowires in a field of view for each treatment. Each data point represents a count for a single field of view, with mean±s.d. indicated ( n =5–7 fields of view for untreated and bare nanowire-treated samples; n =10 fields of view for AIIB2 nanowire-treated samples). (E) Quantification of junction length ratios for each treatment. Each data point represents the average of a single field of view, with mean±s.d. indicated ( n =4 fields of view; 15 measurements per field). Significance determined by two-way repeated measures ANOVA with Bonferroni's correction for multiple comparisons. **** P <0.0001.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Immunofluorescence, Labeling, Quantitation Assay, Competitive Binding Assay, Incubation

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Nanowires differentially affect different claudins. (A–C) Representative immunofluorescence images of Caco-2 cells colabeled to detect ZO-1 (magenta) and either claudin-4 (A), claudin-7 (B) or E-cadherin (C) (all cyan). Cells were fixed 2 h after treatment with either bare, 9EG7 or AIIB2 nanowires (nw). Arrowheads show the ruffled appearance of claudin-4 colocalizing with ZO-1; arrows indicate areas of claudin-7 (B) and E-cadherin (C) that do not colocalize with ruffled ZO-1. Scale bars: 10 μm. Images are representative of three independent experiments.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Immunofluorescence

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Nanowires alter epithelial barrier function. (A) TER measurements on Caco-2 cells for 2 h following treatment as indicated. Measurements were taken every 10 min with a cellZscope impedance system, and all points were normalized to baseline TER readings before treatment. Each point is the average TER for that treatment±s.d. ( n wells/treatment: AIIB2, n =9; 9EG7, n =7; bare, n =6; untreated, n =10). **** P <0.0001 (all treatments were significantly different from the others). (B) Dye flux permeability, as assayed using calcein (630 Da), for Caco-2 cells with the indicated treatments. Each point is the average permeability for that treatment±s.d. ( n wells/treatment: AIIB2, n =7; 9EG7, n =8; bare, n =7; untreated, n =5). * P =0.017 (untreated versus AIIB2 nanowires), **** P <0.0001 (untreated versus 9EG7 nanowires). (C) Dye flux permeability, as assayed using whole fluorescently tagged IgG (160 kDa), for Caco-2 cells with the indicated treatments. Each point is the average permeability for that treatment±s.d. ( n wells/treatment: AIIB2, n =6; 9EG7, n =4; bare, n =4; untreated, n =6). ** P =0.016 (untreated versus AIIB2 nanowires), ** P =0.013 (untreated versus bare nanowires), **** P <0.0001 (AIIB2 nanowires versus bare nanowires and 9EG7 nanowires). In each case, significance was determined by two-way ANOVA with Tukey correction for multiple comparisons.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Permeability

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Talin reorganization in response to nanowires. Representative immunofluorescence images of talin (magenta) and claudin-4 (cyan) in Caco-2 cells fixed 2 h after the indicated nanowire (nw) treatment. Representative line scans as indicated in the merged images were measured using ImageJ to indicate colocalization of talin and claudin-4 in cells treated with bare nanowires (A,B), 9EG7 nanowires (C,D) or AIIB2 nanowires (E,F). Cells treated with bare nanowires or 9EG7 nanowires showed good concordance between talin and claudin-4; however, the distribution of junction-associated talin did not match the distribution of claudin-4 in cells treated with AIIB2 nanowires. Scale bars: 10 μm. Images are representative of two independent experiments.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Immunofluorescence

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Actin reorganization in response to nanowires. (A) Representative fluorescence images of F-actin (labeled using rhodamine–phalloidin) in Caco-2 cells fixed 2 h after the indicated nanowire (nw) treatment. Scale bar: 10 μm. (B) Fluorescence intensity measurements of cells as in A. Data are presented as mean±s.d. ( n =4 fields of view from three slides per condition). **** P <0.0001 for AIIB2 nanowire-treated cells compared with cells treated with either bare nanowires or 9EG7 nanowires. (C) The ratio of cytosolic (stress fibers) to cortical actin for cells as in A. Data are presented as mean±s.d. calculated from 10 cells in a representative field, showing less cytosolic actin for AIIB2 nanowire-treated cells. ** P =0.0018, * P =0.025. Significance determined by one-way ANOVA with Bonferroni correction for multiple comparisons. (D) Caco-2 cells were pre-incubated with either ML-7 (10 μM), Y-27632 (10 μM), PP2 (10 μM), SP-600125 (1 μM) or vehicle control for 30 min followed by 2 h treatment with AIIB2 nanowires, except for the untreated group, which was not exposed to nanowires or inhibitors. Cells were then fixed, immunostained for ZO-1, and imaged. Representative images of ZO-1 immunostaining are shown. Scale bar: 10 μm. (E) Junction length ratios were calculated for cells as in D. Data are presented as mean±s.d. [ n =3 fields of view from one slide (Y-27632) or two slides per condition; 15 measurements per field of view]. ML-7 and Y-27632 significantly inhibited the effects of A2BII nanowires on junction length ratio (** P =0.0011; * P =0.013). Untreated cells had significantly lower junction length ratio than control AIIB2 nanowire-treated cells (*** P <0.0002). Significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons (ns, not significant).

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Fluorescence, Labeling, Incubation, Control, Immunostaining

Journal: Journal of Cell Science

Article Title: Apical integrins as a switchable target to regulate the epithelial barrier

doi: 10.1242/jcs.263580

Figure Lengend Snippet: Model of the impact of anti-integrin nanowires on organization of tight junction proteins and actin. In this model, 9EG7 activating nanowires cluster integrin β1 to promote recruitment of actin, ZO-1 and talin to sites where tight junctions form, increasing their function. By contrast, AIIB2 blocking nanowires decrease actin and talin recruitment, which causes claudins to separate into distinct pools, increasing tight junction permeability.

Article Snippet: Nanowires were conjugated with either AIIB2 blocking anti-integrin β1 antibody (Millipore, MABT409), mAb13 blocking anti-integrin β1 antibody (Millipore, MABT821), 9EG7 activating anti-integrin β1 antibody (BD Pharmingen, 553715), 12G10 activating anti-integrin β1 antibody (Millipore, MAB2247) or an isotype control antibody (Thermo Fisher Scientific, 31933).

Techniques: Blocking Assay, Permeability

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Laminin Modulates the Stem Cell Population in LM05-E Murine Breast Cancer Cells through the Activation of the MAPK/ERK Pathway

doi: 10.4143/crt.2016.378

Figure Lengend Snippet: Laminin (LN) induces tamoxifen resistance in LM05-E cells through integrin α6. (A) LM05-E cells were treated with 4-OH-tamoxifen (TAM) in the presence of laminin (2 μg/mL) for 48 hours, after which cell death was measured as described in the “Materials and Methods.” (B) Whole cell lysates were prepared from LM05-E cells, resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted with α6 integrin antibody. (C) LM05-E cells were treated with TAM for 48 hours in the absence or presence of LN (2 μg/mL) and/or the GoH3 α6 integrin blocking antibody, after which the percentage of dead cells was determined. (D) LM05-E cells were treated for 48 hours with TAM in the absence or presence of LN (2 μg/mL) and/or the AIIB2 β1 integrin blocking antibody and the percentage of dead cells was determined. One of at least three experiments is shown in each case (**p < 0.01, ***p < 0.001).

Article Snippet: The same procedure was used for the AIIB2 β1 integrin blocking antibody (0.15 μg/mL, Aragen Bioscience, Morgan Hill, CA) as previously described [ ].

Techniques: Polyacrylamide Gel Electrophoresis, Blocking Assay

Journal: Nature Communications

Article Title: Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

doi: 10.1038/ncomms15374

Figure Lengend Snippet: ( a – c ) ECs increase the levels of active β1 integrin in hfRPE basal plasma membrane. ( a , b ) Active β1 integrin (blue) and the TJ protein ZO-1 (red) in hfRPE were assessed by immunofluorescence assays (10 z-stacks from 2 biological replicates, t -test). Top panel, orthogonal view (xz) of a confocal z-stack. Bottom panel, confocal plane (xy) indicated by the arrowhead in the top panel. Bar, 10 μm. ( c ) Example of the relative fluorescence of individual confocal planes from 5 z-stacks is shown from the most apical (left) to the most basal (right) planes. ( d ) Inhibition of hfRPE β1 integrin with a blocking antibody impairs EC-mediated increase in TER. For clarity, only the statistical analyses at day 7 are shown ( n =6, ANOVA), all groups versus VeraVec ECs+anti-β1 integrin). ( e , f ) RhoA/ROCK and Rac1 pathways are involved in the EC-mediated increase in hfRPE TER. For clarity in e , only the statistical analyses at week 2 are shown. In e : *Mock control versus mock+ROCK inhibitor; && VeraVec ECs control versus VeraVec ECs+Rac1 inhibitor ( n =6, ANOVA). In f : ***Mock control versus mock+RhoA activator; &&& VeraVec ECs control versus VeraVec ECs+RhoA activator ( n =3, ANOVA). ( g , h ) Cell surface biotinylation assays showing EC-mediated increase in occludin cell surface localization in hfRPE ( n =4, t -test). ( i , j ) Immunofluorescence assays show a lysyl oxidase activity-dependent enhancement of occludin accumulation (green) along ZO-1-positive TJs (red) in hfRPE when exposed to choroid EC-conditioned media ( n =5, ANOVA). Bar, 20 μm. ( k ) Rac1 inhibition impairs EC-mediated occludin accumulation at hfRPE TJs [10 (mock and VeraVec ECs) or 12 (VeraVec ECs+Rac1 inhibitor) z-stacks from 2 biological replicates, ANOVA). ( l , m ) Increasing substrate stiffness enhances occludin localization at hfRPE TJs (10 z-stacks from 2 biological replicates, ANOVA). Bar, 10 μm. ( n ) Inhibiton of lysyl oxidase acitivity does not impair EC-mediated accumulation of collagen I in hfRPE basement membrane (40 images per condition from 2 biological replicates, ANOVA). Data in c – f , h and j are presented as mean±s.d.

Article Snippet: Where indicated, medium in the bottom chamber of Transwell inserts was supplemented with 8 μg ml −1 control rat IgG1 (clone HRPN, cat. MABF1786) or blocking anti-β1 integrin antibody (clone AIIB2 (ref. ), cat. MABT409) (EMD Millipore).

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Fluorescence, Inhibition, Blocking Assay, Control, Activity Assay