Journal: Materials Today Bio

Article Title: Chorion-derived extracellular matrix hydrogel and fibronectin surface coatings show similar beneficial effects on endothelialization of expanded polytetrafluorethylene vascular grafts

doi: 10.1016/j.mtbio.2022.100262

Figure Lengend Snippet: Cytocompatibility, proliferation and adhesion of endothelial cells on coated ePTFE grafts. An increased number of viable cells was observed on hpcECM-gel coated grafts (Calcein AM/Propidium Iodide staining) after 24 ​h (A), but no significant changes in cell viability (B) or proliferation rates (C) were observed over a time period up to 96 ​h. HUVEC adhesion was significantly upregulated in cells seeded on hpcECM-gel compared to fibronectin or non-coated surfaces after 10 ​min of seeding time (D). Proliferation rates of cells which showed higher attachment in the adhesion assay however do not proliferate faster on hpcECM-gel compared to the other conditions (E). An increased number of EPCs were found on hpcECM-gel coated ePTFE grafts after one week of incubation (Prom-1 staining) (F). Scale bars ​= ​50 ​μm, n ​= ​6.

Article Snippet: After a 1 h blocking period with 5% milk in Tris buffered saline with 0.1% Tween20 (TBST), membranes were incubated with primary rabbit anti-human antibodies anti-α5-, anti-β1- and anti-β3 antibodies (1:1000 dilution, Integrin Antibody Sampler Kit, CST, USA) overnight at 4 °C on a roller shaker.

Techniques: Staining, Cell Adhesion Assay, Incubation

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 specifically binds to integrin α3β1-expressing NSCLC cells and their derived exosomes. Chemical structures of LXY30-FITC ( a ) and scrambled-LXY30 (S-LXY30)-FITC ( b ) are illustrated. A549 cells growing in DMEM medium were collected and incubated with LXY30-FITC or S-LXY30-FITC for 2 h, and the binding specificity of LXY30 was evaluated by flow cytometry ( c ) and fluorescence microscopy ( d ). Exosomes were isolated from the supernatant of two integrin α3β1-expressing NSCLC cells (A549 and H1975) and a patient’s malignant pericardial effusion ( e ). Exosomes were incubated with TentaGel bead coated with LXY30 (left panel) or S-LXY30 (right panel) for 2 h, followed by incubation with Alexa 647 mouse anti-CD63 antibody (red) for 1 h before being visualized by fluorescence microscopy ( e ). The expression of integrin α3, integrin β1, and exosomal marker CD63 in 4 NSCLC cell lines and one patient pericardial effusion were determined by Western blots ( f ). Beta-actin was used as loading control for cellular protein expression. The protein loading for cell lysate is around 5 times lower than that of exosomes. h, hour(s); FITC, fluorescein isothiocyanate; NSCLC, non-small cell lung carcinoma

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Derivative Assay, Incubation, Binding Assay, Flow Cytometry, Fluorescence, Microscopy, Isolation, Marker, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Erlotinib-resistant R#2 cells have increased integrin ligands binding compared to their parental erlotinib-sensitive, EGFR-mutant H3255 cells. H3255 (EGFR L585R) and its derived erlotinib-resistant R#2 cells were incubated with different integrin ligands (LXY30, LXW64, and LLP2A) for 2 h, 5 h, or overnight. The binding of tumor cells to integrin ligand-coated beads was visualized by light microscopy, and the intensity of binding was scored from negative (−), weakly, to strongly positive (1+ to 4+) ( a ). The binding of integrin ligands against tumor cells was confirmed by flow cytometry ( b ) and summarized in the table ( c ). h, hour(s); O/N, overnight; EGFR, epidermal growth factor receptor

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Binding Assay, Mutagenesis, Derivative Assay, Incubation, Light Microscopy, Flow Cytometry

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 activates EGFR and its downstream signaling molecules independently from another integrin ligand LXW64. Schematic representation for the cross talk between integrin and EGFR is illustrated ( a ). The effect of LXY30 and/or LXW64 on the expression of integrin subtypes, EGFR, and its key downstream signaling molecules in H1975 ( EGFR L858R/T790 M) cells was analyzed after incubating with LXY30-biotin, LXW64-biotin, or LXY30-biotin/LXW64-biotin for 72 h by Western blot ( b ) and subsequently quantified ( c ). * p < 0.01

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Western Blot

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Integrin expression in tumor cells and tumor-derived exosomes isolated from NSCLC patients

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, Isolation, Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: Optical images showing preferential uptakes of LXY30-biotin-streptavidin-Cy5.5 conjugate (i.e., drug surrogate) in EGFR-resistant lung cancer xenografts in nude mice. After conjugated with streptavidin-Cy5.5, LXY30 shows the capability to deliver imaging dye to subcutaneous xenografts of H1975 ( a , c ) and A549 ( b , d ). Representative images of tumor sections were stained by H&E staining ( e , f ), fluorescent (Alexa 594)-labeled anti-α3 integrin antibody ( g , h ), and integrin α3 IHC ( i , j ). * p < 0.05

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Imaging, Staining, Labeling

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: High α3 integrin expression level is associated with poor prognosis in NSCLC patients. The transcriptome expression data from lung cancer patients were analyzed in The Cancer Genome Atlas (TCGA) database ( http://www.cancergenome.nih.gov ) including 517 lung adenocarcinoma (LUAD) and 502 lung squamous cell carcinoma (LUSC) samples. a The RNA expression levels of α3 integrin (INTA3) gene in LUAD and LUSC, respectively. Kaplan-Meier curves of overall survival were stratified by ITGA3 expression in LUAD ( b ) and LUSC ( c )

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Expressing, RNA Expression

Journal: Journal of Hematology & Oncology

Article Title: High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer

doi: 10.1186/s13045-019-0740-7

Figure Lengend Snippet: LXY30 specifically targets subcutaneous PDX xenograft tumors. A lung squamous cell (LUSC) PDX model was generated by engrafting biopsy specimen into NSG mice as described in the “ ” section. Optical images by white light (WL) and in vivo fluorescence images ( a ) were taken 6 h after the injection of streptavidin-Cy5.5 (SA-Cy5.5) either alone or together with biotinylated LXY30 (LXY30-biotin) into NSG mice bearing subcutaneous LUSC PDX xenografts. Major organs from the NSG mice were subjected to ex vivo imaging immediately after the mice were sacrificed ( b ). The fluorescence signals of SA-Cy5.5 and LXY30-Biotin-SA-Cy5.5 were further quantitated for subcutaneous LUSC PDX tumors ( c ). Representative images of LUSC PDX tumor sections were stained by fluorescent (Alexa594)-labeled anti-α3 integrin antibody, H&E, integrin α3, and integrin β1 immunohistochemistry stains ( d ). PDX, patient-derived xenograft; LUSC, lung squamous carcinoma; WL, white light

Article Snippet: Thirty micrograms of lysates or exosome samples was separated by electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies at 1:400 dilution: pEGFR Y1068, EGFR, pAKT S473, AKT (40D4), pMEK1/2 S217/221, MEK1/2 47E6, pSTAT3 Y705, and STAT3 124H6 (all from Cell Signaling Technology, Danvers, MA); CD63 (Santa sc-365604), integrin α3 (sc-374242), integrin β1 (sc-59829), integrin αV (sc-9969), and β-actin (sc-47778) (Santa Cruz Biotech).

Techniques: Generated, In Vivo, Fluorescence, Injection, Ex Vivo, Imaging, Staining, Labeling, Immunohistochemistry, Derivative Assay