pip3 phosphatidylinositol 3 4 5 trisphosphate  (Echelon Biosciences)

Journal: Nutrients

Article Title: Cell Membrane Fatty Acids and PIPs Modulate the Etiology of Pancreatic Cancer by Regulating AKT

doi: 10.3390/nu17010150

Figure Lengend Snippet: Dietary PUFAs modify the PIP2/PIP3 ratio in the membrane affecting AKT signaling. ( A ) Representative PIP3 IHC images of EK mice pancreata fed normal, ω3-, or ω6-enriched diets (n = 4). ( B ) PIP3 expression score from 0 to 3+, with 0 as no detectable immunostaining, 1 as 10–30% immunostaining, 2 as 30–60%, and 3 as >60%. The numerical score represents the average of 2 independent investigators. ( C ) Representative images of PIP3 immunocytochemical staining of PDAC cell lines incubated with DHA or LA at 40 µM for 48 h. PIP3 immunostaining score of ( D ) Panc-1, ( E ) MiaPaca-2, and ( F ) AsPC-1 cells. Staining was scored from 0 to 3+, with 0 as no detectable immunostaining, 1 as 10–30% immunostaining, 2 as 30–60%, and 3 as >60%. ( G ) Representative images of PIP3 immunocytochemical staining of the Panc-1 cell line incubated with BSA (left image), DHA 40 µM combined with 1 µM of exogenous PIP3 (middle image), and LA 40 µM combined with 1 µM of exogenous PIP2 (n = 3). ( H ) Western blot images of the Panc-1 pAKT levels after the exogenous supplementation of PIP2 and PIP3 to the PUFAs treatment. Results are expressed as the mean ± standard deviation (SD). Asterisks above a group define significance against the corresponding untreated control: * p < 0.05; ** p < 0.005; *** p < 0.0005. Results are expressed as mean ± standard deviation (SD).

Article Snippet: For the exogenous supplementation of phosphoinositide, PIP2 (phosphatidylinositol (4,5)-bisphosphate and PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) were purchased from Echelon Biosciences Inc. and administered to the cells (at 1 µM) also conjugated with BSA in the same conditions described above.

Techniques: Membrane, Expressing, Immunostaining, Staining, Incubation, Western Blot, Standard Deviation, Control

Journal: Nutrients

Article Title: Cell Membrane Fatty Acids and PIPs Modulate the Etiology of Pancreatic Cancer by Regulating AKT

doi: 10.3390/nu17010150

Figure Lengend Snippet: Dietary PUFAs modify PIP3 localization in the membrane, affecting AKT signaling, and exogenous PUFA administration by lipid nanoparticles (LNPs) improves PUFA delivery to the membranes. ( A ) Representative images of Panc-1 cells expressing the PH-BtK-EGF fusion protein (PIP3 biosensor) and incubated with 40 µM of DHA and LA. GFP (PIP3) expression was assessed with a confocal microscope. White arrows point to GFP-enriched spots at the plasma membrane. ( B ) Percentage of cells with membrane-positive staining relative to the total number of green cells (4 different fields of view, 20×). ( C ) Panc-1 cells transfected with GFP-C1-PLCdelta-PH were incubated with DHA and LA 40 µM for 48 h and subjected to immunoprecipitation. Western blot images of PI3k-alpha pulled down with GFP antibody to assess the binding of PI3K to PIP3. ( D ) Quantification of the replicates performed by immunoblotting. Quantification was done using ImageJ (NIH) software. All the images are representative of the averaged results of the scoring (n = 2). ( E ) The Panc-1 cell line was incubated with a lipid nanoparticle formulation (LNP) consisting of 90% GMO and 10% cholesterol with 40 µM DHA. The BSA group represents the control group treated with DHA bound to BSA (BSA.DHA) as a carrier. The LNP group represents the group treated with DHA encapsulated in LNP (LNP-DHA). Western blot images probing for total and phosphorylated AKT proteins. GAPDH was used as a loading control. ( F ) Quantification of the replicates performed by immunoblotting relative to the GAPDH expression level. ( G ) Representative images of confocal microscopy of Panc-1 cells treated with the complex BSA-DHA and with the complex LNP-DHA. Fatty acids are shown in red, LNP are shown in green, and the nucleus in blue. Asterisks in the graphs above a group define significance against the normal diet control: * p < 0.05; *** p < 0.0005. Bars indicate significant differences between two groups. Results are expressed as the mean ± standard deviation (SD).

Article Snippet: For the exogenous supplementation of phosphoinositide, PIP2 (phosphatidylinositol (4,5)-bisphosphate and PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) were purchased from Echelon Biosciences Inc. and administered to the cells (at 1 µM) also conjugated with BSA in the same conditions described above.

Techniques: Membrane, Expressing, Incubation, Microscopy, Staining, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Software, Formulation, Control, Confocal Microscopy, Standard Deviation