phosphatidylinositol 3 4 5 trisphosphate pip3  (Echelon Biosciences)

Journal: Frontiers in Genetics

Article Title: A PIK3R2 Mutation in Familial Temporal Lobe Epilepsy as a Possible Pathogenic Variant

doi: 10.3389/fgene.2021.596709

Figure Lengend Snippet: PIP3 expression levels in iPSCs derived from the proband and a healthy control. (A,B) Immunofluorescence staining of PIP3 in iPSCs derived from the proband (A) and the healthy control (B) . Images were taken with the same exposure time. Scale bar corresponds to 20 μm. (C) Elevated PIP3 expression is evident in iPSCs derived from the proband. The average PIP3 level among the cells is based on the mean fluorescence intensity, which is calculated with ImageJ software. A two-tailed t -test was used to compare the mean fluorescence intensity. ** indicates a significant statistical difference ( p < 0.01). Error bar indicates SEM.

Article Snippet: Then, the cells were incubated with an anti-phosphatidylinositol-3,4,5-trisphosphate (PIP3) monoclonal antibody (1:50; MBL, Japan; D145-3) overnight at 4°C, followed by a goat anti-mouse IgG (Alexa Fluor® 594) (1:800; Abcam, United Kingdom; ab150116) secondary antibody for 1 h at room temperature.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Software, Two Tailed Test

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

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

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