Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Immunoblot analysis of mouse CMT-93 cells infected with M1 wt, and recombinant M1-ΔE1A-G and M1-IX-G viruses using an MOI of 3. Cell lysate samples were harvested at six time points and analyzed with the indicated rabbit antibodies raised against early E1A-M1, E1B-19K-M1, intermediate protein IX-M1, and the late hexon protein-M1, plus mouse antibodies against GFP and control actin. Staining with protein IX-specific antibodies revealed a weak band corresponding to processed IX-2A (Mr 14.1 kDa), and a major form corresponding to unprocessed IX-2A-GFP (Mr 41 kDa). Staining with GFP-specific antibodies revealed two major processing forms, corresponding to processed GFP (Mr 27 kDa), and the unprocessed IX-2A-GFP, respectively. Both of these stainings gave rise to additional individual protein forms (denoted by *). (B) Mouse CMT-93 cells and (C) human M000216 cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber-chimeric H5-ΔE3B-CG-FK-M1 and–FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (upper panel) and percent infected cells (lower panel) were determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. Data represent triplicates, shown as mean ± SEM.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Western Blot, Infection, Recombinant, Staining, Flow Cytometry

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A-D) CMT-93 cells were incubated for 1 h on ice using 5-fold dilution series of the indicated FK proteins starting with 0.8 μg/ml as highest concentration, followed by addition of the different GFP-expressing viruses and transfer to 37°C for 48 h. The virus input amounted to an MOI of 1 and included M1-IX-G (A), M3-IX-G (B), H5-ΔE3B-CG-FK-M1 (C), M2-ΔE1A-G (D). GFP analysis was performed 48 h pi, and expression index was normalized to FK-H3 control protein. (E) M000216 cells were preincubated and processed as described for CMT-93 cells, except that M1-IX-G was used at an MOI of 3. (F) The M3-IX-G and H5-ΔE3B-CG-FK-M3 viruses were pre-incubated for 1 h at RT with serial 5-fold dilutions of the different antisera ranging from 1:1,250 to 1:781,250, followed by addition of the mixes to CMT-93 cells for 48 h at 37°C. The rabbit antisera tested were raised against recombinant FK-M3, FK-M2 and FK-H3, respectively. The virus input amounted to an MOI of 1, and samples were processed for analysis as described above. (G) To check for cross-neutralization of the rabbit anti-FK-M3 for M1, M1-IX-G was preincubated with serial dilutions of rabbit anti-FK-M3, -FK-M2 and -FK-H3 sera and further processed as described above. For all experiments, data represent triplicates, shown as mean ± SEM. For highest concentrations of FKs and anti-FK sera, asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005); ns: not significant ( P >0.05).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Incubation, Concentration Assay, Expressing, Virus, Recombinant, Neutralization, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) The green and brown histograms show cytofluorometric analysis of FLAG-tag expression levels in parental B16 and B16-mCAR cells, consisting of B16 cells stably expressing N-terminal FLAG-tagged mCAR, respectively. The grey histogram shows background staining of B16-mCAR cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (B) Parental B16 cells with known low, and CMT-93 cells with high sensitivity for H5-ΔE1-CG, plus B16-mCAR cells were infected with M1-IX-G, M2-ΔE1A-G, M3-IX-G and H5-ΔE1-CG using an MOI of 15 for both B16 cell types, and an MOI of 3 for CMT-93 cells. Cells were analyzed by flow cytometry 48 h pi. MFI values for GFP in red and blue histograms are from uninfected and infected cells, respectively.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: FLAG-tag, Expressing, Stable Transfection, Staining, Infection, Flow Cytometry

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Flow Cytometry, Staining, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Comparison

Journal: PLOS ONE

Article Title: An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi -Non Toxic

doi: 10.1371/journal.pone.0289183

Figure Lengend Snippet: (A) Pictural description of the α V β 3 adhesion assay methodology. (B) Fold change observed in crystal violet (CV) absorbance at a wavelength of 590 for wild type (WT), non-toxic (NT) C . novyi as well as the putative RGD-modified candidates (A and B) after exposure to the α V β 3 coated surface of the adhesion assay. * denotes a p value of < 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. (C) Average CV pixel count of entire integrin coated surface for candidates A and B as well as wild-type (WT) and non-toxic (NT) C . novyi that remain on the α V β 3 coated surface. ‡ denotes a p value of 0.05 when compared to any other cohort, including WT, NT, and modification Candidate B. Error bars represent standard deviation from the cumulative mean of three experimental replications (n = 6 each) for a total n = 18.

Article Snippet: Purified α V β 3 integrin (100μL of 10μg/mL carrier-free, human recombinant protein, R&D Systems Bio-Techne 3050-AV) solution was administered to the center of the corona-treated cover slips.

Techniques: Cell Adhesion Assay, Modification, Standard Deviation

Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: (A) Representative MR images from mice bearing 4T1‐GFP macrometastases at Days 28–35. (Ai,ii) Tumours imaged with RGD‐MPIO; (Ai) Nonenhancing tumour at Day 28, (Aii) Gadolinium‐enhancing tumour at Day 35. (Aiii–iv) Tumours imaged with RDG‐MPIO; (Aiii) Nonenhancing tumour at Day 28, (Aiv) Gadolinium‐enhancing tumour at Day 35. Each column of images, from the left, shows T 1 ‐weighted postgadolinium images, T 2 *‐weighted pre‐MPIO MGE3D images, T 2 *‐weighted post‐MPIO MGE3D images, and overlays on T 2 *‐weighted MGE3D images showing hypointensities pre‐MPIO and post‐MPIO. For the overlays, the tumour‐bearing striatum is segmented in green, and the contralateral striatum segmented in pink. Hypointense voxels are shown in red. (B) Significantly increased RGD‐MPIO–induced hypointense voxels were evident in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (C) Significantly increased control RDG‐MPIO–induced hypointense voxels were also seen in the tumour‐bearing striatum (white bars) compared with the contralateral striatum (black bars) at Day 35 (two‐way paired ANOVA, p < 0.05). (D–E) Comparison of pooled data across all timepoints for (D) Nonenhancing tumours, and (E) Gadolinium‐enhancing tumours. (D) Mice with nonenhancing tumours administered RGD‐MPIO (white bars; n = 7) showed significantly increased MPIO‐induced hypointense voxels in the tumour‐bearing hemisphere (one‐way ANOVA, p < 0.005) than both the contralateral hemisphere and the mice administered control RDG‐MPIO (black bars; n = 13) in the tumour‐bearing hemisphere. (E) In mice with gadolinium‐enhancing tumours, significantly increased MPIO‐induced hypointense voxels were observed in the tumour‐bearing striatum compared with the contralateral striatum for both RGD‐MPIO (white bars; n = 10) and RDG‐MPIO (black bars; n = 3) (one‐way ANOVA, p < 0.0001). However, in mice receiving control RDG‐MPIO, the volume of MPIO‐induced hypointense voxels was also significantly greater than those administered RGD‐MPIO. Number of MPIO‐induced hypointense voxels, presented as postcontrast minus precontrast hypointense voxels for all data. Bars represent mean ± standard deviation; post‐hoc Holm–Sidak's tests. * p < 0.05, *** p < 0.001. MGE3D, multigradient echo three‐dimensional; MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Control, Comparison, Standard Deviation

Journal: Nmr in Biomedicine

Article Title: Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using α v β 3 ‐targeted iron oxide microparticles

doi: 10.1002/nbm.4948

Figure Lengend Snippet: Aggregations of iron in 4T1‐GFP tumour tissue. (A) Representative section of gadolinium‐enhancing tumour stained for Perls' Prussian blue (iron, blue) and counterstained with nuclear fast red. Black arrowheads indicate examples of single MPIO associated with the vascular endothelium, and red arrowheads indicate examples of iron aggregations. (B–C) Correlations between iron aggregations and tumour size in mice injected with either (B) RGD‐MPIO or (C) Control RDG‐MPIO. Linear regression analysis showed a positive correlation between number of aggregations and tumour size ( R 2 = 0.64, *** p < 0.001) in mice injected with RGD‐MPIO, but not control RDG‐MPIO, although a similar trend was evident; 95% confidence intervals are shown. (D–F) Consecutive sections stained for (D) Iba‐1 (macrophages and microglia, brown staining), (E) Prussian blue (iron), and (F) CD31 (blood vessels, brown staining) in a Day 35 mouse injected with RGD‐MPIO. The red arrow indicates a larger iron aggregation in a similar location to macrophage staining (D; Iba‐1), while the black arrow indicates single MPIO distant from macrophage staining, but close alignment with a blood vessel (F; CD31). (G–H) Double staining of 4T1‐GFP tumour tissue sections, from a mouse injected with RGD‐MPIO at the Day 35 timepoint, for Prussian blue (iron) and macrophages/microglia (Iba‐1, brown staining), indicate colocalisation of iron within macrophages/microglia (red arrows). Sections counterstained with nuclear fast red. (I) Double staining for Prussian Blue and CD31 revealed single MPIO (black arrow) bound to blood vessels (brown stained) in mice injected with RGD‐MPIO; representative image from Day 21 shown. (J) In the gadolinium‐enhancing 4T1‐GFP tumours, single endothelium‐bound MPIO are observed more often in mice injected with RGD‐MPIO ( n = 10) than control RDG‐MPIO ( n = 3, t ‐test, ** p < 0.01). (K) Histogram showing the cumulative frequency of the measured distance between the centre of the iron‐laden macrophages and the centre of the blood vessel lumen, indicating their close association with blood vessels. Scale bar = 25 μm in (A) and 10 μm in (D–I). MPIO, microparticles of iron oxide; RDG, Arg‐Asp‐Gly peptide, scrambled control; RGD, Arg‐Gly‐Asp peptide, targeting integrin α v β 3 .

Article Snippet: Subsequently, capillaries were filled with 200 ng/mL mouse integrin α v β 3 (7889‐AV‐050, R&D Systems, Abingdon, UK) and incubated at room temperature for 24 h. The remaining functional groups were quenched with 10 mM ethanolamine for 24 h at room temperature.

Techniques: Staining, Injection, Control, Double Staining

Journal: American Journal of Translational Research

Article Title: Lyn kinase enhanced hepatic fibrosis by modulating the activation of hepatic stellate cells

doi:

Figure Lengend Snippet: Increased activation of HSCs in Lyn TG mice upon CCl4 treatment. A: Representative immunofluorescence photomicrographs of HSC activation marker (Αvβ3, α-SMA and desmin) expression in liver tissues from WT mice and Lyn TG mice after administration of CCl4 (×200 magnification). B: The fluorescence intensity of desmin in 8 random fields. C: The fluorescence intensity of α-SMA in 8 random fields. D: The fluorescence intensity of Αvβ3 in 8 random fields. E: TGF-β1 levels in the liver tissues were determined by ELISA. All of the data are presented as the mean ± s.d. One-way ANOVA (Tukey-Kramer post-test) was used for Statistical analysis.

Article Snippet: After blocking with 1% BSA (Sigma-Aldrich) containing 0.05% Tween 20 for 1 hour, the specimens were then incubated with anti-Lyn antibody (Santa Cruz, CA, sc-15, AB_2281450), anti-integrin αvβ3 antibody (Bioss, China, bs-1310R), anti-α-SMA antibody (Bioss, China, bs-0189R), anti-desmin antibody (Bioss, China, bs-1026R), and anti-COL1α1 antibody (Bioss, China, bs-10423R).

Techniques: Activation Assay, Immunofluorescence, Marker, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Translational Research

Article Title: Lyn kinase enhanced hepatic fibrosis by modulating the activation of hepatic stellate cells

doi:

Figure Lengend Snippet: Lyn kinase increased the activation of HSCs in vitro. Immunohistochemical staining for HSC activation marker (Αvβ3, α-SMA and COL1α1) expression in TGF-β1-induced LX-2 and Lyn+/+ LX-2. A: Representative photomicrographs and fluorescence intensity of α-SMA in cells (×400 magnification). B: Representative photomicrographs and fluorescence intensity of Αvβ3 in cells (×400 magnification). C: Representative photomicrographs and fluorescence intensity of COL1α1 in cells (×400 magnification). All of the data are presented as the mean ± s.d. One-way ANOVA (Tukey-Kramer post-test) was used for Statistical analysis. LX-2 cell lines transfected with lentiviral vector expressing Lyn were described as Lyn+/+ cells. LX-2 cell lines transfected with control lentiviral vector were described as WT cells.

Article Snippet: After blocking with 1% BSA (Sigma-Aldrich) containing 0.05% Tween 20 for 1 hour, the specimens were then incubated with anti-Lyn antibody (Santa Cruz, CA, sc-15, AB_2281450), anti-integrin αvβ3 antibody (Bioss, China, bs-1310R), anti-α-SMA antibody (Bioss, China, bs-0189R), anti-desmin antibody (Bioss, China, bs-1026R), and anti-COL1α1 antibody (Bioss, China, bs-10423R).

Techniques: Activation Assay, In Vitro, Immunohistochemical staining, Staining, Marker, Expressing, Fluorescence, Transfection, Plasmid Preparation

Journal: American Journal of Translational Research

Article Title: Lyn kinase enhanced hepatic fibrosis by modulating the activation of hepatic stellate cells

doi:

Figure Lengend Snippet: The Src-specific inhibitor PP2 suppressed HSC activation. A: Immunohistochemical staining for HSC activation marker (Αvβ3) expression in TGF-β1-induced LX-2 and Lyn+/+ LX-2 in the presence of PP2 (×400 magnification). The fluorescence intensity of Αvβ3 in cells was measured. B: Apoptotic cells of LX-2 were detected by flow cytometry. All of the data are presented as the mean ± s.d. One-way ANOVA (Tukey-Kramer post-test) was used for Statistical analysis.

Article Snippet: After blocking with 1% BSA (Sigma-Aldrich) containing 0.05% Tween 20 for 1 hour, the specimens were then incubated with anti-Lyn antibody (Santa Cruz, CA, sc-15, AB_2281450), anti-integrin αvβ3 antibody (Bioss, China, bs-1310R), anti-α-SMA antibody (Bioss, China, bs-0189R), anti-desmin antibody (Bioss, China, bs-1026R), and anti-COL1α1 antibody (Bioss, China, bs-10423R).

Techniques: Activation Assay, Immunohistochemical staining, Staining, Marker, Expressing, Fluorescence, Flow Cytometry

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti-avβ3 integrin, DAB chromogen, magnification x 200, score 0 in a case of unexplained infertility group

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti-avβ3 integrin, DAB chromogen, magnification x 200, score 3 in a case of fertility group. Reactivity is mainly in the galndular epithelium)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti- vβ3 integrin, DAB chromogen, magnification x 40 (Low Power), score 3 in A case of fertility group. Reactivity could be seen in both Luminal Epithelium (LE) the Glandular Epithelium (GE)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Immunostained slide with anti- vβ3 integrin, DAB chromogen, magnification x 200 (High Power), score 3 in A case of fertility group. Reactivity could be seen Mainly in Luminal Epithelium (red arrow) rather than the Glandular Epithelium (red star)

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Comparison of endometrial thickness, subendometrial Doppler resistance index, and ανβ3-integrin score in both study groups

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Comparison, Control

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Receiver-operating characteristic (ROC) curve for the discrimination between patients with unexplained infertility and normal controls using a : Endometrial thickness, b : Subendometril RI, c : avβ3 integrin

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques:

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Receiver-operating characteristic (ROC) curve analysis for the discrimination between patients with unexplained infertility and control group

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Control

Journal: BMC Women's Health

Article Title: AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

doi: 10.1186/s12905-017-0438-3

Figure Lengend Snippet: Summary of studies looked at endometrial αVβ3 integrin in luteal phase of infertile women

Article Snippet: After blocking with non-immune 4% goat serum incubated with tissues for 30 min, the primary antibody using monoclonal mouse IgG antibodies directed against human αvβ3 integrin (Human integrin alphaV beta3 MAb (clone 23C6)®, MAB3050, R&D systems, Minneapolis, USA) was added to these sections.

Techniques: Control, Expressing