shuttle ptdins 4 p kit  (Echelon Biosciences)


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    Echelon Biosciences shuttle ptdins 4 p kit
    Shuttle Ptdins 4 P Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shuttle ptdins 4 p kit  (Echelon Biosciences)


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    Echelon Biosciences shuttle ptdins 4 p kit
    Shuttle Ptdins 4 P Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptdins 3 4 5 p3  (Echelon Biosciences)


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    Echelon Biosciences ptdins 3 4 5 p3
    Ptdins 3 4 5 P3, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptdins  (Echelon Biosciences)


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    Echelon Biosciences ptdins
    Ptdins, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pip 2 shuttle kit  (Echelon Biosciences)


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    Echelon Biosciences pip 2 shuttle kit
    A–D The level of <t>PIP</t> <t>2</t> in M‐MOPS was examined by measuring immunofluorescence from images at set time points after exposure to 5 µg/ml anti‐FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1–42 (A). The levels of PIP 2 were also measured using a plate reader after exposure to physiologically relevant stimuli in M‐MOPS and primary mouse microglia (B). DAG level was measured using a live‐cell assay in M‐MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522). See also Appendix Fig .
    Pip 2 Shuttle Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522"

    Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020105603

    A–D The level of PIP 2 in M‐MOPS was examined by measuring immunofluorescence from images at set time points after exposure to 5 µg/ml anti‐FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1–42 (A). The levels of PIP 2 were also measured using a plate reader after exposure to physiologically relevant stimuli in M‐MOPS and primary mouse microglia (B). DAG level was measured using a live‐cell assay in M‐MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522). See also Appendix Fig .
    Figure Legend Snippet: A–D The level of PIP 2 in M‐MOPS was examined by measuring immunofluorescence from images at set time points after exposure to 5 µg/ml anti‐FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1–42 (A). The levels of PIP 2 were also measured using a plate reader after exposure to physiologically relevant stimuli in M‐MOPS and primary mouse microglia (B). DAG level was measured using a live‐cell assay in M‐MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522). See also Appendix Fig .

    Techniques Used: Immunofluorescence

    A–C Iba1 was used as a marker for microglia, and PIP 2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data show the mean ± SD of three independent experiments and were analysed using two‐way ANOVA with Sidak’s multiple comparison tests performed. ** P < 0.01 and **** P < 0.0001. See also Appendix Fig .
    Figure Legend Snippet: A–C Iba1 was used as a marker for microglia, and PIP 2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data show the mean ± SD of three independent experiments and were analysed using two‐way ANOVA with Sidak’s multiple comparison tests performed. ** P < 0.01 and **** P < 0.0001. See also Appendix Fig .

    Techniques Used: Marker, Fluorescence

    pi 4 5 p2 liposomes  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p2 liposomes
    Pi 4 5 P2 Liposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    5 bisphosphate pip2  (Echelon Biosciences)


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    Echelon Biosciences 5 bisphosphate pip2
    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, <t>PIP2</t> and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )
    5 Bisphosphate Pip2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition"

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    Journal: Cell Research

    doi: 10.1038/s41422-019-0229-5

    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )
    Figure Legend Snippet: PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Techniques Used: Imaging, Mutagenesis

    PIP3 exhaustion governs VEGFA-induced regression of CDS2-deficient endothelium. Confocal images ( a ) and quantitative analysis ( b ) of vessel-deficient phenotype in WT zebrafish embryos or cds2 mutants with or w/o vegfa OE, with control MO or pten MO (combination of ptena and ptenb MO) injection. The counted ISV number shown on the top ( b ) is from 15–20 embryos per group. c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or pten MO. The expression was normalized to WT embryos. n = 3 samples per group, 15–20 embryos pooled for each sample. d pten knockdown partially restored PIP3, but not PIP2 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples each group. Confocal images ( e and f ) and quantitative analysis ( g ) of P7 retinal vessels stained by IB4 ( e ) and IB4/COL4 ( f ) from control or VEGFA-injected Cds2 iΔEC mice treated with bpV (PTEN inhibitor) or vehicle (saline). bpV (2 mg kg −1 ) or vehicle was given three times at P2, P4 and P6. Arrows show regressed vessels. Angiogenic sprouts, COL4 + /IB4 − empty sleeves, endothelial area and branch points were quantified in ( g ), n = 6–10 mice per group. Scale bars, 100 μm ( a and f ) and 200 μm ( e ). Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.
    Figure Legend Snippet: PIP3 exhaustion governs VEGFA-induced regression of CDS2-deficient endothelium. Confocal images ( a ) and quantitative analysis ( b ) of vessel-deficient phenotype in WT zebrafish embryos or cds2 mutants with or w/o vegfa OE, with control MO or pten MO (combination of ptena and ptenb MO) injection. The counted ISV number shown on the top ( b ) is from 15–20 embryos per group. c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or pten MO. The expression was normalized to WT embryos. n = 3 samples per group, 15–20 embryos pooled for each sample. d pten knockdown partially restored PIP3, but not PIP2 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples each group. Confocal images ( e and f ) and quantitative analysis ( g ) of P7 retinal vessels stained by IB4 ( e ) and IB4/COL4 ( f ) from control or VEGFA-injected Cds2 iΔEC mice treated with bpV (PTEN inhibitor) or vehicle (saline). bpV (2 mg kg −1 ) or vehicle was given three times at P2, P4 and P6. Arrows show regressed vessels. Angiogenic sprouts, COL4 + /IB4 − empty sleeves, endothelial area and branch points were quantified in ( g ), n = 6–10 mice per group. Scale bars, 100 μm ( a and f ) and 200 μm ( e ). Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Techniques Used: Injection, Expressing, Western Blot, Quantitation Assay, Staining

    PIP3 reduction is mainly caused by PLCγ mediated PIP2 hydrolysis. Representative confocal images ( a ) and quantitative analysis ( b ) of trunk vessel phenotypes of WT or cds2 mutant embryos with vegfa OE and with or w/o plcg1 MO. The ISV number counted from 20–22 embryos per group is shown on the top ( b ). c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or plcg1 MO. vegfa expression level was determined at 2 h post heatshock induction and normalized to WT embryos without vegfa OE. n = 3 samples, 15–20 embryos pooled for each sample. d plcg1 knockdown partially restored PIP2 and PIP3 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples per group. e Working model of VEGFA-triggered vessel regression on CDS2-deficient endothelium. The outcome of VEGFA signaling can be reversed from angiogenesis to vessel regression, which is dependent on CDS2-controlled PIP2 and PIP3 availability and FOXO1 signaling activation. Scale bar, 100 μm. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.
    Figure Legend Snippet: PIP3 reduction is mainly caused by PLCγ mediated PIP2 hydrolysis. Representative confocal images ( a ) and quantitative analysis ( b ) of trunk vessel phenotypes of WT or cds2 mutant embryos with vegfa OE and with or w/o plcg1 MO. The ISV number counted from 20–22 embryos per group is shown on the top ( b ). c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or plcg1 MO. vegfa expression level was determined at 2 h post heatshock induction and normalized to WT embryos without vegfa OE. n = 3 samples, 15–20 embryos pooled for each sample. d plcg1 knockdown partially restored PIP2 and PIP3 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples per group. e Working model of VEGFA-triggered vessel regression on CDS2-deficient endothelium. The outcome of VEGFA signaling can be reversed from angiogenesis to vessel regression, which is dependent on CDS2-controlled PIP2 and PIP3 availability and FOXO1 signaling activation. Scale bar, 100 μm. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Techniques Used: Mutagenesis, Injection, Expressing, Western Blot, Quantitation Assay, Activation Assay

    phosphatidylinositol 4  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 4
    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )
    Phosphatidylinositol 4, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition"

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    Journal: Cell Research

    doi: 10.1038/s41422-019-0229-5

    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )
    Figure Legend Snippet: PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Techniques Used: Imaging, Mutagenesis

    ptdins 4 5 p2 shuttle pip kit p 9045  (Echelon Biosciences)


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    Echelon Biosciences ptdins 4 5 p2 shuttle pip kit p 9045
    Ptdins 4 5 P2 Shuttle Pip Kit P 9045, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi 3 4 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 3 4 p 2
    Pi 3 4 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pip 3  (Echelon Biosciences)


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    Echelon Biosciences pip 3
    Pip 3, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pip 2 shuttle kit
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    Image Search Results


    A–D The level of PIP 2 in M‐MOPS was examined by measuring immunofluorescence from images at set time points after exposure to 5 µg/ml anti‐FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1–42 (A). The levels of PIP 2 were also measured using a plate reader after exposure to physiologically relevant stimuli in M‐MOPS and primary mouse microglia (B). DAG level was measured using a live‐cell assay in M‐MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522). See also Appendix Fig .

    Journal: The EMBO Journal

    Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

    doi: 10.15252/embj.2020105603

    Figure Lengend Snippet: A–D The level of PIP 2 in M‐MOPS was examined by measuring immunofluorescence from images at set time points after exposure to 5 µg/ml anti‐FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1–42 (A). The levels of PIP 2 were also measured using a plate reader after exposure to physiologically relevant stimuli in M‐MOPS and primary mouse microglia (B). DAG level was measured using a live‐cell assay in M‐MOPS (and primary mouse microglia as a time course from immunofluorescence (C) and plate reader after exposure to physiologically relevant stimuli (D). Data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522). See also Appendix Fig .

    Article Snippet: Hyperloading of PIP 2 into mouse macrophage cells was achieved using a PIP 2 shuttle kit (Echelon Biosciences P‐9045), as per the manufacturer’s instructions.

    Techniques: Immunofluorescence

    A–C Iba1 was used as a marker for microglia, and PIP 2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data show the mean ± SD of three independent experiments and were analysed using two‐way ANOVA with Sidak’s multiple comparison tests performed. ** P < 0.01 and **** P < 0.0001. See also Appendix Fig .

    Journal: The EMBO Journal

    Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

    doi: 10.15252/embj.2020105603

    Figure Lengend Snippet: A–C Iba1 was used as a marker for microglia, and PIP 2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data show the mean ± SD of three independent experiments and were analysed using two‐way ANOVA with Sidak’s multiple comparison tests performed. ** P < 0.01 and **** P < 0.0001. See also Appendix Fig .

    Article Snippet: Hyperloading of PIP 2 into mouse macrophage cells was achieved using a PIP 2 shuttle kit (Echelon Biosciences P‐9045), as per the manufacturer’s instructions.

    Techniques: Marker, Fluorescence

    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Journal: Cell Research

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    doi: 10.1038/s41422-019-0229-5

    Figure Lengend Snippet: PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

    Techniques: Imaging, Mutagenesis

    PIP3 exhaustion governs VEGFA-induced regression of CDS2-deficient endothelium. Confocal images ( a ) and quantitative analysis ( b ) of vessel-deficient phenotype in WT zebrafish embryos or cds2 mutants with or w/o vegfa OE, with control MO or pten MO (combination of ptena and ptenb MO) injection. The counted ISV number shown on the top ( b ) is from 15–20 embryos per group. c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or pten MO. The expression was normalized to WT embryos. n = 3 samples per group, 15–20 embryos pooled for each sample. d pten knockdown partially restored PIP3, but not PIP2 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples each group. Confocal images ( e and f ) and quantitative analysis ( g ) of P7 retinal vessels stained by IB4 ( e ) and IB4/COL4 ( f ) from control or VEGFA-injected Cds2 iΔEC mice treated with bpV (PTEN inhibitor) or vehicle (saline). bpV (2 mg kg −1 ) or vehicle was given three times at P2, P4 and P6. Arrows show regressed vessels. Angiogenic sprouts, COL4 + /IB4 − empty sleeves, endothelial area and branch points were quantified in ( g ), n = 6–10 mice per group. Scale bars, 100 μm ( a and f ) and 200 μm ( e ). Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Journal: Cell Research

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    doi: 10.1038/s41422-019-0229-5

    Figure Lengend Snippet: PIP3 exhaustion governs VEGFA-induced regression of CDS2-deficient endothelium. Confocal images ( a ) and quantitative analysis ( b ) of vessel-deficient phenotype in WT zebrafish embryos or cds2 mutants with or w/o vegfa OE, with control MO or pten MO (combination of ptena and ptenb MO) injection. The counted ISV number shown on the top ( b ) is from 15–20 embryos per group. c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or pten MO. The expression was normalized to WT embryos. n = 3 samples per group, 15–20 embryos pooled for each sample. d pten knockdown partially restored PIP3, but not PIP2 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples each group. Confocal images ( e and f ) and quantitative analysis ( g ) of P7 retinal vessels stained by IB4 ( e ) and IB4/COL4 ( f ) from control or VEGFA-injected Cds2 iΔEC mice treated with bpV (PTEN inhibitor) or vehicle (saline). bpV (2 mg kg −1 ) or vehicle was given three times at P2, P4 and P6. Arrows show regressed vessels. Angiogenic sprouts, COL4 + /IB4 − empty sleeves, endothelial area and branch points were quantified in ( g ), n = 6–10 mice per group. Scale bars, 100 μm ( a and f ) and 200 μm ( e ). Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

    Techniques: Injection, Expressing, Western Blot, Quantitation Assay, Staining

    PIP3 reduction is mainly caused by PLCγ mediated PIP2 hydrolysis. Representative confocal images ( a ) and quantitative analysis ( b ) of trunk vessel phenotypes of WT or cds2 mutant embryos with vegfa OE and with or w/o plcg1 MO. The ISV number counted from 20–22 embryos per group is shown on the top ( b ). c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or plcg1 MO. vegfa expression level was determined at 2 h post heatshock induction and normalized to WT embryos without vegfa OE. n = 3 samples, 15–20 embryos pooled for each sample. d plcg1 knockdown partially restored PIP2 and PIP3 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples per group. e Working model of VEGFA-triggered vessel regression on CDS2-deficient endothelium. The outcome of VEGFA signaling can be reversed from angiogenesis to vessel regression, which is dependent on CDS2-controlled PIP2 and PIP3 availability and FOXO1 signaling activation. Scale bar, 100 μm. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Journal: Cell Research

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    doi: 10.1038/s41422-019-0229-5

    Figure Lengend Snippet: PIP3 reduction is mainly caused by PLCγ mediated PIP2 hydrolysis. Representative confocal images ( a ) and quantitative analysis ( b ) of trunk vessel phenotypes of WT or cds2 mutant embryos with vegfa OE and with or w/o plcg1 MO. The ISV number counted from 20–22 embryos per group is shown on the top ( b ). c Relative vegfa mRNA level of cds2 mutants with vegfa OE injected with ctrl or plcg1 MO. vegfa expression level was determined at 2 h post heatshock induction and normalized to WT embryos without vegfa OE. n = 3 samples, 15–20 embryos pooled for each sample. d plcg1 knockdown partially restored PIP2 and PIP3 level in cds2 -deficient endothelium with vegfa OE. Western blotting analysis on a-Tubulin serves as the internal control for cell amounts in each sample collected during lipid quantitation analysis. n = 4 samples per group. e Working model of VEGFA-triggered vessel regression on CDS2-deficient endothelium. The outcome of VEGFA signaling can be reversed from angiogenesis to vessel regression, which is dependent on CDS2-controlled PIP2 and PIP3 availability and FOXO1 signaling activation. Scale bar, 100 μm. Error bars, mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P ≥ 0.05). See also Supplementary information, Fig.

    Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

    Techniques: Mutagenesis, Injection, Expressing, Western Blot, Quantitation Assay, Activation Assay

    PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Journal: Cell Research

    Article Title: Endothelial CDS2 deficiency causes VEGFA-mediated vascular regression and tumor inhibition

    doi: 10.1038/s41422-019-0229-5

    Figure Lengend Snippet: PI-PIPs metabolism is involved in CDS2-deficient vessel regression. a Schematic diagram of CDS2-controlled phosphoinositide recycling. DAG, diacylglycerol; PA, phosphatidic acid; PIS1, phosphatidylinositol synthase 1; PI, phosphoinositol; PIK, phosphoinositol 3/4/5-kinase; PIPK, phosphatidylinositol 4/5-phosphate 5/4-kinase; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3 kinase; PLC, phospholipase c; PG, phosphatidylglycerol; CL, cardiolipin. b Model of timing for phospholipid-carrier mixture microinjection, heatshock induction and confocal imaging analysis in ( c ) and ( d ). c Quantification of rescue effects of different phospholipids (PI, PG, PIP2 and PIP3) on vegfa OE-induced ISV regression in cds2 mutant embryos. Bars show percentages of vessel deficiency. The representative images of classified vascular phenotype are shown on the right. The counted ISV numbers were shown on the top, 10 ISV per embryo. d Representative images of trunk vessels from cds2 mutants at 76–80 hpf with or w/o vegfa OE and with microinjection of lipid–carrier complex, including PG, PI, PIP2 or PIP3 at 28–30 hpf. Scale bars, 50 μm ( c ) and 100 μm ( d )

    Article Snippet: For phospholipid delivery, phosphatidylinositol (PI) (Echelon Biosciences, P-0004; 1 mg ml −1 ), phosphatidylglycerol (PG) (Sigma, P8318; 2.5 mg ml −1 ), phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (Echelon Biosciences, P-3904; 1 mg ml −1 ) and phosphatidylinositol 4, 5-bisphosphate (PIP2) (Echelon Biosciences, P-9045; 1 mg ml −1 ) were dissolved in water and incubated with equal molar concentration of histone H1 carrier (Echelon Biosciences, P-9C2) for 10 min at room temperature before injection.

    Techniques: Imaging, Mutagenesis