Journal: International Journal of Molecular Sciences

Article Title: Intracellular Ca 2+ Increases and Connexin 43 Hemichannel Opening Are Necessary but Not Sufficient for Thy-1-Induced Astrocyte Migration

doi: 10.3390/ijms19082179

Figure Lengend Snippet: Expression levels of β3 integrin increase at the cell surface and form β3 integrin receptor clusters. ( A ) Analysis of β3 integrin abundance at the cell surface by flow cytometry (FACS) analysis performed in non-permeabilized primary astrocytes. The graph depicts the mean fluorescence intensity in astrocytes in the absence and the presence of TNF. Values in the graph are mean ± s.e.m. of five independent experiments per condition. * p < 0.05; ( B ) The histogram shows a comparison of β3 integrin expression levels in cells that were within the gates of the control cells, cells without TNF or with TNF, from one representative experiment. Control condition was carried out in the absence of the primary antibody (gray line). Results correspond to 10,000 events per sample; ( C ) Confocal images of non-treated primary astrocytes or cells treated with TNF (48 h) in the presence or absence of Thy-1 (15 min). Non-permeabilized cells were stained for αvβ3 integrin (red) and DAPI (blue), which was used to stain the nuclei. The merge of both colors is also shown. Scale bar = 5 µm.

Article Snippet: Astrocytes were stained with anti-αvβ3 integrin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) followed by goat anti-mouse IgG conjugated to Alexa fluor 546 (Molecular Probes, Eugene, OR, USA); and DAPI (diamidino-2-phenylindole) (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Fluorescence, Comparison, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Intracellular Ca 2+ Increases and Connexin 43 Hemichannel Opening Are Necessary but Not Sufficient for Thy-1-Induced Astrocyte Migration

doi: 10.3390/ijms19082179

Figure Lengend Snippet: β3 integrin overexpression forms integrin clusters at the cell surface. ( A ) Cell lysates of primary astrocytes transfected with pEGFP or pEGFP-β3 integrin were immunoblotted for β3 integrin and β-actin as a loading control; ( B ) Confocal images of primary astrocytes transfected with pEGFP, pEGFP-GPI or pEGFP-β3 integrin were acquired 24 h after plasmid transfection. Scale bar = 20 µm. Outlined area defines the optically zoomed region shown below each photograph (bar = 10 µm); ( C ) Dye uptake by primary astrocytes transfected as in A, stimulated with Thy-1-Fc or ionomicyn (1 µM) for 10 min, or treated with the intracellular calcium chelator BAPTA-AM (5 µM) for 30 min prior to stimuli. Values in the graph are the mean fluorescence intensity normalized to the non-stimulated condition. Results show mean ± s.e.m. from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Astrocytes were stained with anti-αvβ3 integrin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) followed by goat anti-mouse IgG conjugated to Alexa fluor 546 (Molecular Probes, Eugene, OR, USA); and DAPI (diamidino-2-phenylindole) (Sigma-Aldrich).

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Intracellular Ca 2+ Increases and Connexin 43 Hemichannel Opening Are Necessary but Not Sufficient for Thy-1-Induced Astrocyte Migration

doi: 10.3390/ijms19082179

Figure Lengend Snippet: Signaling pathways involved in TNF/Thy-1-induced astrocyte polarization and migration. In astrocytes, TNF treatment induces integrin microcluster formation, increases Cx43 levels at the plasma membrane and prepares cells to respond to Thy-1. Once Thy-1 binds to the integrin microclusters, the receptors oligomerize further, thereby leading to the formation of bigger clusters and integrin activation. The active integrin protein then recruits signaling molecules, such as FAK, PI3K, Src, PLCγ. The activation of the phospholipase generates diacylglycerol (DAG) and IP 3 , which activates the inositol 1,4,5-trisphosphate receptor (IP 3 R) to allow the release of Ca 2+ from intracellular stores. Increased intracellular Ca 2+ leads to the opening of hemichannels, the consequent release of ATP to the extracellular medium, and the activation of P2X7 receptors, which allow Ca 2+ entry into the cell. This signaling pathway is part of a more complex chain of events that eventually leads to cell polarization and migration (modified from ). Arrows with solid lines indicate proven connections which may or may not be direct. Arrows with dashed lines indicate connections with non-specified/multiple in between steps.

Article Snippet: Astrocytes were stained with anti-αvβ3 integrin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) followed by goat anti-mouse IgG conjugated to Alexa fluor 546 (Molecular Probes, Eugene, OR, USA); and DAPI (diamidino-2-phenylindole) (Sigma-Aldrich).

Techniques: Protein-Protein interactions, Migration, Clinical Proteomics, Membrane, Activation Assay, Modification

Journal: bioRxiv

Article Title: Defective intestinal repair after short-term high fat diet due to loss of efferocytosis

doi: 10.1101/790220

Figure Lengend Snippet: ( a ) Quantification of percent of tethered TAMRA positive neutrophils in sorted macrophage from LFD and HFD mice after DSS treatment (LFD DSS (n=4), HFD DSS (n=4)) ( b ) Gene expression of Abca1 in sorted intestinal macrophages (n= 4, 3 mice per group pooled per “n”). ( c ) Flow cytometry analysis for surface expression of αvβ3/5 in intestinal macrophages of LFD and HFD control and DSS treated mice (n= 4 mice per group). ( d ) Gene expression of Cd36 in cecum tissue from LFD and HFD control and DSS treated mice (n= 5 LFD/HFD, n=7 LFD/HFD+DSS per group). ( e ) Flow cytometry analysis for surface expression of CD36 in intestinal macrophages in LFD and HFD control and DSS treated mice (n= 4 mice per group). ( f ) Representative immunofluorescence staining for milk-fat globule-EGF factor 8 (Mfge8, green), nuclei (DAPI, blue) and apoptotic cells (TUNEL, red) in LFD and HFD control and DSS treated mice (n= 5 mice per group). ( g ) Signal ratio of Mfge8 from HFD and LFD treated mice on day 9 post DSS per high-powered field (n=5 mice per group average of 3 images per mouse). Data are presented as mean ± SEM. Statistical significance was analyzed by Student t Test ( a, b, d, e ).

Article Snippet: The following antibodies were used for flow staining and or sorting: MHC II (M5/114.15.2, BioLegend Cat# 107620), CD11b (M1/70, Biolegend Cat# 101226), CD11c (N418, Biolegend, Cat # 117317), Ly6C (1A8, BD PharMingen Cat#560525), CD36 (HM36, Biolegend CA# 102605), αvβ5 (ALULA, BD PharMingen Cat#565836), αvβ3 (RMV-7, BD PharMingen Cat#104106), CD45 (30-F11, Biolegend Cat#103149), CD103 (2E7, BioLegend Cat#121413), DAPI (Sigma-Aldrich Cat# D9542).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining, TUNEL Assay

Journal: Heliyon

Article Title: Fibronectin promotes tumor progression through integrin αvβ3/PI3K/AKT/SOX2 signaling in non-small cell lung cancer

doi: 10.1016/j.heliyon.2023.e20185

Figure Lengend Snippet: FN promotes tumor growth through integrin αvβ3. ( A) The relative expression of integrin αv, αII, α8 and α5 in FN-treated or untreated A549 cells and Lewis cells for 72 h at the mRNA level. (B) The relative gene expression of integrin β3, β5, β6 and β8 in FN-treated or untreated A549 and Lewis cells for 72 h. (C) Western blot of integrin αvβ3 in A549 and Lewis cells treated or untreated with FN for 72 h. (D) Relative cell proliferation of A549 and Lewis cells treated with PBS, FN or FN plus CT for 72 h. (E) The NSCLC tumor weight of mice. A total of 1 × 10 6 A549 and Lewis cells pretreated with PBS or FN or FN plus CT for 72 h were intratracheally instilled into nude mice. Tumor weight was calculated on day 20 and day 15 respectively. (F) Relative colony numbers and representative images of A549 and Lewis cells treated with PBS, FN or FN plus CT for 72 h. The scale bar is 30 μm. (G) The tumorigenesis of nude mice intratracheally instilled with 1 × 10 5 A549 cells and Lewis cells pretreated with PBS, FN or FN plus CT for 72 h. (H) Immunofluorescence of integrin αvβ3 in tumor tissues of high malignant or low malignant NSCLC patients. Scale bar, 20 μm. Error bar, mean ± SEM; * P < 0.05; ** P < 0.01; ns, not statistically significant. Cilengitide, CT.

Article Snippet: The sections were then blocked with 5% BSA and incubated with mouse monoclonal anti-integrin αvβ3 (1:100, ab7166, Abcam, UK), rabbit monoclonal SOX2 (1:200, ab92494, Abcam, Cambridge, UK), rabbit polyclonal p-AKT (1:200, ab38449, Abcam, Cambridge, UK), rabbit monoclonal KLF (1:200, ab215036, Abcam, Cambridge, UK), rabbit monoclonal total-PI3K (1:500, ab302958, Abcam, Cambridge, UK), mouse monoclonal CD90 (1:100, ab181496, Abcam, Cambridge, UK) for 4 °C overnight, and followed by horse radish peroxidase (HRP) conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Heliyon

Article Title: Fibronectin promotes tumor progression through integrin αvβ3/PI3K/AKT/SOX2 signaling in non-small cell lung cancer

doi: 10.1016/j.heliyon.2023.e20185

Figure Lengend Snippet: Combination of integrin αvβ3 inhibitor and chemotherapeutic agents enhances anticancer effects in NSCLC treatment. ( A) The tumor weight analysis of lung tissues from mice on day 35 after intratracheally instillation withA549 cells and treatment with PBS, PTX or PTX combined with CT. (B) The survival time of A549-bearing mice treated with PBS, PTX or PTX combined with CT. (C) The NSCLC tumor weight of mice on day 35 after intratracheally instillation with A549 cells and treatment with PBS, MTX or MTX combined with CT. (D) The survival time of A549-bearing mice treated with PBS, MTX or MTX combined with CT. (E) The NSCLC tumor weight of mice on day 25 after intratracheally instillation with Lewis cells and treatment with PBS, PTX or PTX combined with CT. (F) The survival time of Lewis-bearing mice treated with PBS, PTX or PTX combined with CT. (G) The FN-treated NSCLC tumor weight of mice on day 35 after intratracheally instillation with 1 × 10 6 A549 cells and treatment with PBS, PTX or PTX combined with CT. (H) The survival time of FN-treated A549 bearing mice treated with PBS, PTX or PTX combined with CT. Error bars, mean ± SEM; * P < 0.05; ** P < 0.01; ns, not statistically significant. Cilengitide, CT.

Article Snippet: The sections were then blocked with 5% BSA and incubated with mouse monoclonal anti-integrin αvβ3 (1:100, ab7166, Abcam, UK), rabbit monoclonal SOX2 (1:200, ab92494, Abcam, Cambridge, UK), rabbit polyclonal p-AKT (1:200, ab38449, Abcam, Cambridge, UK), rabbit monoclonal KLF (1:200, ab215036, Abcam, Cambridge, UK), rabbit monoclonal total-PI3K (1:500, ab302958, Abcam, Cambridge, UK), mouse monoclonal CD90 (1:100, ab181496, Abcam, Cambridge, UK) for 4 °C overnight, and followed by horse radish peroxidase (HRP) conjugated secondary antibodies (1:600; Abcam, Cambridge, UK), and the nucleus was stained with DAPI.

Techniques: