human subunit β 5  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: ISM inhibits in vitro angiogenesis through α v β 5 integrin. ( a ) Transient transfection of β 3 and β 5 siRNA effectively knocked down their expression in ECs. Same amount of corresponding scrambled siRNA are transfected as control. ( b ) Only knockdown of β 5-integrin expression abolished the anti-tube formation activity of ISM

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: In Vitro, Transfection, Expressing, Control, Knockdown, Activity Assay

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: ISM induces apoptosis through IMD. ( A ) ISM induced apoptosis through caspase-8-mediated extrinsic pathway, but not caspase-9-mediated intrinsic pathway. ( B ) Kinetics of ISM-induced apoptosis. Activated caspase-8 can first be detected 4 h after ISM treatment. ( C ) Knockdown of β 5-integrin subunit by siRNA suppressed caspase-8 and caspase-3 activation, as well as cleavage of PARP. ( D ) ISM induces caspase-8 association with α v β 5 integrin and activation. ( E ) Soluble ISM induces α v β 5-integrin clustering. Co-localization of β 5 integrin (red) with ISM (green) is indicated by yellow signals. DAPI (blue) stains the nucleus. Scale bar: 20 μ m

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Knockdown, Activation Assay

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: ISM inhibits in vitro angiogenesis through α v β 5 integrin. ( a ) Transient transfection of β 3 and β 5 siRNA effectively knocked down their expression in ECs. Same amount of corresponding scrambled siRNA are transfected as control. ( b ) Only knockdown of β 5-integrin expression abolished the anti-tube formation activity of ISM

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: In Vitro, Transfection, Expressing, Control, Knockdown, Activity Assay

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: ISM induces EC apoptosis through α v β 5 integrin. ( a ) Knockdown of β 5, but not β 3 integrin, suppressed ISM-induced apoptosis. ( b ) Knockdown of β 5 integrin rescued ISM-suppressed proliferation. ( c ) More free α v β 5 integrins are available in ECs growing on FN-coated surface. VN, vitronectin; ISM, isthmin; FN, fibronectin. ( d ) ISM induces more extensive apoptosis in ECs grown on FN-coated surface

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Knockdown

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: ISM induces apoptosis through IMD. ( A ) ISM induced apoptosis through caspase-8-mediated extrinsic pathway, but not caspase-9-mediated intrinsic pathway. ( B ) Kinetics of ISM-induced apoptosis. Activated caspase-8 can first be detected 4 h after ISM treatment. ( C ) Knockdown of β 5-integrin subunit by siRNA suppressed caspase-8 and caspase-3 activation, as well as cleavage of PARP. ( D ) ISM induces caspase-8 association with α v β 5 integrin and activation. ( E ) Soluble ISM induces α v β 5-integrin clustering. Co-localization of β 5 integrin (red) with ISM (green) is indicated by yellow signals. DAPI (blue) stains the nucleus. Scale bar: 20 μ m

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Knockdown, Activation Assay

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: Immobilized ISM promotes EC adhesion through α v β 5 integrin. ( A ) ECs attach to ISM-coated surface in a dose-dependent and saturable manner. ( B ) Similar to VN, ISM supports EC adhesion and spreading, induces integrin clustering and actin stress fiber formation. Cell spreading in the absence or presence of EDTA are shown in phase contrast micrographs (panels A – D , scale bar: 40 μ m). Actin stress fibers (red) and β 5 integrin clustering (green) are shown in panels E–H (scale bar: 20 μ m). ( C ) ISM mediates EC adhesion through α v β 5 integrin. ( D ) Knockdown of β 3 and β 5 integrin suppresses EC adhesion to VN. ( E ) Knockdown of β 5 integrin suppresses EC adhesion to ISM

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Knockdown

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: Immobilized ISM activates FAK and promotes EC haptotactic migration. ( a ) Immobilized ISM activates FAK Y397 phosphorylation in similar fashion as immobilized VN. S, cells in suspension. ( b ) ISM dose-dependently promotes EC haptotactic migration with similar potency as FN. ( c ) ISM promotes EC haptotactic migration through α v β 5 integrin

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Migration, Phospho-proteomics, Suspension

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: Identification of the α v β 5 integrin-binding site in ISM. ( a ) Schematic diagrams showing the truncated ISM protein fragments produced as recombinant proteins in E. coli . Numbers refer to the amino acid position in the full-length secreted ISM protein. The positon of KD316AA mutation is indicated by the arrowhead. The black triangle at the right end of each truncated protein represent His-tag. ( b ) EC adhesion to various ISM protein fragments compared with 0.2% gelatin. 3% BSA was used as the negative control. ( c ) KD316AA mutation abolished the anti-tube formation activity of ISM

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Binding Assay, Produced, Recombinant, Mutagenesis, Negative Control, Activity Assay

Journal: Cell Death & Disease

Article Title: Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

doi: 10.1038/cddis.2011.37

Figure Lengend Snippet: Schematic illustration of the dual functions of ISM. ( a ) Immobilized ISM serves as integrin agonist to mediate cell survival/migration by activating FAK. ( b ) Soluble ISM induces cell apoptosis through IMD (direct activation of caspase-8). ‘?' indicates the unresolved question if additional molecules are involved in the integrin–caspase-8 complex formation

Article Snippet: siRNAs were purchased from 1st BASE (Singapore): siRNA targeting human β 3 integrin subunit mRNA (5′-CAAGCCUGUGUCACCAUAC-dTdT-3′); human β 5 subunit integrin mRNA (5′-GCUCGCAGGUCUCAACAUA-dTdT-3′); and a random sequence control siScramble (5′-GACGUGGGACUGAAGGGGU-dTdT-3′) were as described before.

Techniques: Migration, Activation Assay