Journal: Cell

Article Title: Type V collagen in scar tissue regulates the size of scar after heart injury

doi: 10.1016/j.cell.2020.06.030

Figure Lengend Snippet: (A) Expression of ECM and myofibroblast genes in Col5a1CKO CFs generated ex vivo (n=6) (B,C) Flow cytometry to determine expression of (B) αvβ3 and (C) αvβ5 integrins on Col5a1CKO CFs (n=6). (D,E) Immunostaining for Vim and (D) αvβ3, (E) αvβ5 in scar tissue at 7 days post-MI (arrows, representative images) (F) Expression of key myofibroblast genes in Col5a1CKO CFs in the presence or absence of cilengitide (n=6). (G) Experimental design to treat animals with daily cilengitide (20mg/kg) (H) EF/FS in control and Col5a1CKO injected with cilengitide or vehicle (*Col5a1CKO+Cilengitide [red dotted line] vs. Col5a1CKO+Veh [red solid line], n=13/CKO+Cilengitide 10/other groups at basal, n=12/CKo+Cilengitide, 6/CKO+Veh, 9/Control+Cilengitide, 7/Control+Veh at 2wks post MI). (I) Representative images of M mode echocardiogram (yellow line indicates end systolic diameter) (J) Masson trichrome staining of mid ventricle at 2 weeks post-MI to show scar size (arrowhead, n=same number at 2 weeks post-MI as above) (K) Quantitation of fibrotic area (n=same number as above) (L) Fraction of Col5a1CKO animals demonstrating mild, moderate and severe fibrosis following PBS or cilengitide infusion (M) Immunostaining for αSMA and Vimentin in hearts of Col5a1CKO receiving PBS or cilengitide and quantitation of the fraction (arrows, representative images, n=10/CKO+Cilengitide, n=6/CKO+Veh, n=6/animals for all other groups). (N) Dot plot representing expression of ECM genes that are upregulated in CFs of Col5a1CKO hearts at 7 days post-MI compared to controls (left panel), the same genes were shown in fibroblasts from Col5a1CKO+Vehicle and Col5a1CKO+Cilengitide samples (right panel). (O) Box plot showing the module scores of 28 genes from (N) in fibroblasts from Col5a1CKO+Veh and Col5a1CKO+Cilengitide. Data shown as mean± S.D., *p<0.05, ns: Not significant.

Article Snippet: The following primary antibodies, reagents, or probes were used for immunostaining: rabbit anti-Vimentin (1:100, Abcam, ab45939); mouse anti-smooth muscle actin (1:100, Dako, M0851); anti-cardiac Troponin I (1:100, Abcam, ab47003); mouse anti-integrin αVβ3 (1:50, Abcam, ab7166); mouse integrin αVβ5 (1:20, R&D, MAB2528); Alexa Fluor 594 conjugated WGA (5μg/ml, Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"W11262","term_id":"1285567","term_text":"W11262"}} W11262 ).

Techniques: Expressing, Generated, Ex Vivo, Flow Cytometry, Immunostaining, Injection, Staining, Quantitation Assay

Journal:

Article Title: Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

doi: 10.1093/emboj/cdg416

Figure Lengend Snippet: Fig. 1. OS internalization requires tyrosine kinase activity. OS binding induces tyrosine phosphorylation of αvβ5 complexes. (A) OS binding by RPE-J cells during 2 h of OS incubation was determined in the presence of solvent (white bar), 10 and 50 µM genistein (10, 50, gray bars) or 1–10 µg/ml herbimycin A (1, 5, 10, black bars). αvβ5 antibody P1F6 (50 µg/ml) inhibited OS binding similarly in the presence of solvent (white striped bar, c), 50 µM genistein (gray striped bar, 50) or 10 µg/ml herbimycin (black striped bar, 10). (B) OS internalization by RPE was determined after 1–5 h of OS challenge in the presence of solvent as control (×), genistein [10 µM (open circle), 50 µM (closed circle)], or 5 µg/ml herbimycin A (closed triangle). (A and B) show average OS indices ± SD (n = 3). (C) Tyrosine-phosphorylated proteins in RPE were detected by immunofluorescence staining after 1 h of challenge with medium (medium), OS (OS), OS in the presence of 50 µM genistein (OS + G), or OS in the presence of 5 µg/ml herbimycin A (OS + H). Three-dimensional (3D) projections are shown. Scale bars: 10 µm. (D) Immunoblotting was used to detect tyrosine-phosphorylated proteins in immunoprecipitates with non-immune IgG (–) and αvβ5 (+) IgG (panel PY). Reprobing of blots with αv antibody showed similar amounts of precipitated αvβ5 (panels αv). RPE cells were lysed after 1 h of challenge with OS (OS) or medium (m) in the absence or presence of 5 µg/ml herbimycin A as indicated (m + H, OS + H). The open arrowhead indicates the molecular size of FAK.

Article Snippet: Immunoprecipitation of αvβ5 integrin with 2.5 µg of P1F6 αvβ5 heterodimer-specific antibody (Covance, Richmond, CA) was performed exactly as described previously ( Finnemann and Rodriguez-Boulan, 1999 ).

Techniques: Activity Assay, Binding Assay, Incubation, Immunofluorescence, Staining, Western Blot

Journal:

Article Title: Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

doi: 10.1093/emboj/cdg416

Figure Lengend Snippet: Fig. 2. FAK localizes to the apical, phagocytic RPE surface. OS uptake controls FAK complex formation with αvβ5 and its phosphorylation at Tyr397 in the complex. (A) Polarized RPE-J cells were labeled with FAK and ZO-1 tight-junction antibodies. Confocal x–y scans were acquired at 2 µm intervals starting at the basal surface (bottom) as indicated. Scale bar: 10 µm. (B) z-axis confocal scan of the same samples further reveals apical localization of FAK. (A and B) show one representative field each. (C) RPE lysates were harvested after 1 and 3 h of incubation with OS (OS) or with medium (m). Immunoblotting served to detect FAK in immunoprecipitations with non-immune (–) and αvβ5 (+) IgG. (D) The same (–) and (+) immuno precipitations were performed after 1 and 3 h of OS challenge (OS) before immunoblotting to detect complexed FAK-P-Tyr397. Lower panels in (C) and (D) show blot reprobed with αv antibody. (E) FAK and αv band intensities were compared in immunoprecipitates to quantify FAK–αvβ5 complexes. (F) FAK-P-Tyr397 and αv band intensities were compared in immunoprecipitates to quantify FAK-P-Tyr397–αvβ5 complexes. Bars in (E and F) represent averages ± SD, n = 4.

Article Snippet: Immunoprecipitation of αvβ5 integrin with 2.5 µg of P1F6 αvβ5 heterodimer-specific antibody (Covance, Richmond, CA) was performed exactly as described previously ( Finnemann and Rodriguez-Boulan, 1999 ).

Techniques: Labeling, Incubation, Western Blot

Journal:

Article Title: Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

doi: 10.1093/emboj/cdg416

Figure Lengend Snippet: Fig. 6. Inhibition of FAK signaling blocks internalization but not binding of OS. RPE-J cells were infected with FRNK (F) or GFP (c) adenovirus. (A) Fluorescence scanning was used to establish OS binding indices after 2 h of OS challenge (gray bars) and OS internalization indices after 5 h of OS challenge (black bars). Bars show averages ± SD, n = 4. (B) Immunoblotting with FAK C-terminus antibody detected full-length endogenous FAK (FAK) and exogenous FRNK (FRNK) in whole-cell lysates (total), and in soluble (s) and insoluble (i) fractions prepared by differential detergent extraction. (C) Bars show the relative amount of insoluble FAK. Bars represent averages ± SD, n = 3. (D) Full-length FAK was detected in αvβ5 integrin immunoprecipitations. (E) Bars show average amounts of αvβ5-complexed FAK ± SD, n = 3. (F) Whole-cell lysates of untreated cells (/) or cells receiving medium (m) or OS (OS) for 3 h were immunoblotted for FAK-P-Tyr397 and for total FAK as indicated. (G) Bars show quantification of FAK-P-Tyr397 compared with total FAK within each sample. Bars represent averages ± SD, n = 3. (H) Immunoprecipitates with non-immune IgG (–) or Crk antibody (+) were isolated from lysates of untreated cells (/) or cells receiving medium (m) or OS (OS) for 3 h. Immunoblotting with p130CAS antibody detected co-precipitated p130CAS (1st WB:p130CAS). Blots were stripped and re-probed with CrkII antibodies (2nd WB:CrkII). (I) Bars show relative amounts of p130CAS complexed with Crk. Bars represent averages ± SD, n = 3.

Article Snippet: Immunoprecipitation of αvβ5 integrin with 2.5 µg of P1F6 αvβ5 heterodimer-specific antibody (Covance, Richmond, CA) was performed exactly as described previously ( Finnemann and Rodriguez-Boulan, 1999 ).

Techniques: Inhibition, Binding Assay, Infection, Fluorescence, Western Blot, Isolation

Journal:

Article Title: Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

doi: 10.1093/emboj/cdg416

Figure Lengend Snippet: Fig. 8. αvβ5 ligation is sufficient to phosphorylate and mobilize FAK. RPE-J cells received assay medium (med), OS (OS), αvβ5 receptor mouse IgG P1F6 (P1F6), non-immune mouse IgG (n.i. IgG), P1F6 plus crosslinking anti-mouse IgG antibodies (P1F6/X) or non-immune IgG plus crosslinking antibodies (n.i. IgG/X) for 3 h (A and B) or 1–5 h as indicated (C) before lysis. Experiments were performed in the presence or absence of 4% FCS as indicated. (A) Immunoprecipitations with non-immune IgG (–) or αvβ5 IgG (+) were probed with FAK-antibody detecting FAK at 120 kDa (FAK) and with αv-antibody (αv) detecting αv at 140 kDa. (B) The same experiment as in (A) was performed in the absence of serum. (C) Identical whole-cell lysate immunoblots were probed with FAK-P-Tyr397 and FAK C-terminus antibodies to determine FAK autophosphorylation.

Article Snippet: Immunoprecipitation of αvβ5 integrin with 2.5 µg of P1F6 αvβ5 heterodimer-specific antibody (Covance, Richmond, CA) was performed exactly as described previously ( Finnemann and Rodriguez-Boulan, 1999 ).

Techniques: Ligation, Lysis, Western Blot

Journal:

Article Title: Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors

doi: 10.1093/emboj/cdg416

Figure Lengend Snippet: Fig. 9. FAK signaling is upstream of and required for MerTK phosphorylation. (A) Long Evans or RCS RPE received OS for 2 h in the presence of 50 µg/ml non-immune (n.i. Ab) or αvβ5 antibody (αvβ5 Ab) before labeling of surface-bound OS and counting of bound (black bars) and internal (gray bars) OS. Bars show averages ± SD, n = 4. (B) Comparative immunoblotting of lysates as indicated for each panel showed increased FAK phosphorylation at Tyr397 in response to OS (OS) but not to medium (m) over untreated controls (/) in Long Evans as well as in RCS RPE. Bars show average FAK-P-Tyr397 levels relative to untreated RPE ± SD, n = 3. (C) RPE-J cells received medium (3 h medium: MerTK) or Cy5-labeled OS (3 h OS panels) for 3 h before fixation and labeling of MerTK. 3D projections representing apical cell domains acquired as described in Figure 4 are shown. Scale bars: 10 µm. (D) RPE-J cells expressing FRNK, as characterized in Figure 6, received medium (m) or (OS) before lysis and immuno precipitation of MerTK. Immunoprecipitates were blotted and probed first with phosphotyrosine antibody (panel IP:MerTK WB:PY) and reprobed with MerTK antibody (panel IP:MerTK WB:MerTK). Expression of transfected GFP and FRNK as well as full-length FAK and actin as controls in cell lysates are also shown, as indicated in the panels. Bars show average MerTK tyrosine phosphorylation ± SD, n = 3. FRNK significantly inhibited OS-induced MerTK tyrosine phosphorylation with P < 0.01.

Article Snippet: Immunoprecipitation of αvβ5 integrin with 2.5 µg of P1F6 αvβ5 heterodimer-specific antibody (Covance, Richmond, CA) was performed exactly as described previously ( Finnemann and Rodriguez-Boulan, 1999 ).

Techniques: Labeling, Western Blot, Expressing, Lysis, Immunoprecipitation, Transfection

Journal: British Journal of Cancer

Article Title: Binding of TGF-β1 latency-associated peptide (LAP) to αvβ6 integrin modulates behaviour of squamous carcinoma cells

doi: 10.1038/sj.bjc.6600545

Figure Lengend Snippet: Cell adhesion to LAP is αvβ6-dependent. Chromium [ 51 Cr]- labelled cells (1.5×10 4 ) were added to LAP-coated 96-well plates containing an irrelevant control antibody (W632 anti-MHC class 1) or test antibodies against αv (L230), αvβ5 (P1F6), αvβ6 (10D5), β1 (P4C10) and α5β1 (P1D6). Background binding to BSA has been subtracted from the results. Figures show representative experiments performed in quadruplicate. Error bars represent standard deviation. ( A ) VB6 cells show increased adherence LAP. β6 transfected cells (VB6), control cells (C1) and αv-negative cells (H357) were plated onto varying concentrations of LAP. VB6 cells showed significantly increased adhesion compared to C1 cells (which express low levels of endogenous αvβ6). H357 cells did not adhere. ( B ) Adhesion to LAP is αvβ6-dependent. VB6 and C1 adhesion to LAP was inhibited by anti-αvβ6 antibody or anti-αv antibody. Antibodies against αvβ5, α5β1 or β1 produced no effect. These data suggest that adhesion of VB6 and C1 cells is modulated solely through αvβ6.

Article Snippet: Anti-αvβ5 (P1F6) was obtained from Life Technologies, Paisley, UK.

Techniques: Binding Assay, Standard Deviation, Transfection, Produced

Journal: British Journal of Cancer

Article Title: Binding of TGF-β1 latency-associated peptide (LAP) to αvβ6 integrin modulates behaviour of squamous carcinoma cells

doi: 10.1038/sj.bjc.6600545

Figure Lengend Snippet: Cell migration towards LAP is αvβ6-dependent. Cells were allowed to migrate towards LAP in haptotactic migration assays. To assess integrin specificity of migration, integrin-blocking antibodies against αv (L230), αvβ5 (P1F6), αvβ6 (10D5), or a control antibody (W632) were added to VB6, C1 and H357 cells prior to plating into wells. Following 8 h incubation, the cells in the lower chamber (including those attached to the undersurface of the membrane) were trypsinised and counted on a Casy 1 counter (Sharfe System GmbH, Germany). Results for the cell lines are expressed relative to VB6 migration following incubation with an irrelevant control antibody (=100). Figures show representative experiments performed in quadruplicate. Error bars represent standard deviation. ( A ) VB6 cells show increased migration towards LAP. Comparison of migration of VB6, C1 and H357 cells towards LAP. Migration is higher in the αvβ6 expressing VB6 cells. αv-negative H357 cells did not migrate significantly. ( B ) Migration towards LAP is αvβ6-dependent. Migration of VB6 and C1 cells was inhibited completely by antibody inhibition of αvβ6 or αv. Antibodies inhibiting αvβ5 produced no effect. These data suggest that migration of VB6 and C1 cells is modulated solely through αvβ6.

Article Snippet: Anti-αvβ5 (P1F6) was obtained from Life Technologies, Paisley, UK.

Techniques: Migration, Blocking Assay, Incubation, Standard Deviation, Expressing, Inhibition, Produced

Journal: BMC Cancer

Article Title: The fibronectin III-1 domain activates a PI3-Kinase/Akt signaling pathway leading to αvβ5 integrin activation and TRAIL resistance in human lung cancer cells

doi: 10.1186/s12885-016-2621-6

Figure Lengend Snippet: FnIII-1c stimulates the PI3K-Akt-dependent activation of the αvβ5 integrin. a NCI-H460 cells were seeded in complete medium for 48 h and then immunostained (FITC) for the indicated integrins. Positive staining was seen only for αvβ5 integrin. Panel A' shows αvβ5 integrins in focal contacts of individual cells. Bar = 10 μm. Nuclei were counterstained with Hoechst 33342. b NCI-H460 cells were pretreated with 10 μM FnIII-1c or PBS for 1 h before seeding onto wells coated with the designated concentrations of vitronectin. After 1 h, cell adhesion was measured by toluidine staining. The data are presented as the mean absorbance (OD) ± SE of at least 3 independent experiments. Results were analyzed by a two-way Anova with Tukey’s post-hoc analysis (*, p < 0.05). c H460 cell monolayers were incubated with blocking antibody to the αvβ3 (LM609), αvβ5 (P1F6) integrin or control mouse IgG. Cells were then treated with 10 μM FnIII-1c for 1 h prior to stimulation with 100 ng/ml TRAIL for an additional 2.5 h. Lysates were immunoblotted for cleaved caspase 8 and GAPDH. Cells treated with PBS or FnIII-1c served as additional controls. A representative blot is shown. d Densitometric quantification of the data shown in ( c ). Data are presented as the amount of cleaved caspase 8 relative to TRAIL-treated cells, which was set at 1. Each bar represents the mean ± SE of three independent experiments. A one-way ANOVA with Tukey post-hoc analysis was used to determine statistical significance (*, p < 0.05). e NCI-H460 cells were pre-treated with 10 μM VIII (Akt1/2 kinase inhibitor) or 10 μM LY294002 (PI3K inhibitor) for 30 min before treatment with FnIII-1c. Cells treated with PBS served as control. Treated cells were seeded onto plates coated with vitronectin (0.5 μg/ml) and allowed to adhere for 1 h. Adhesion was measured by toluidine staining (OD) and presented as fold-change relative to FnIII-1c-treated cells, which was set at 1. The data represent the mean ± SE of three independent experiments. Results were analyzed by a two-way Anova with Sidak’s multiple comparison tests (*, p < 0.05)

Article Snippet: Mouse monoclonal antibodies against integrin αvβ5 (15 F11), integrin β3 (LM609), integrin αv (MAB1980), α5β1, and the monoclonal blocking antibody against integrin αvβ5 (P1F6) were purchased from Millipore (Billerica, MA).

Techniques: Activation Assay, Staining, Incubation, Blocking Assay