Journal: bioRxiv

Article Title: Epinephrine inhibits PI3K alpha via the Hippo kinases

doi: 10.1101/2022.07.19.500601

Figure Lengend Snippet: (A) Experimental schema. (B) Dendrogram of the human protein kinome that highlights the kinases selected for our screen. (C) Thin layer chromatography (TLC)-autoradiography of [ 32 P]PIP produced by recombinant PI3Kα complexes (p110α/p85α, 500 nM) after incubation with the indicated recombinant protein kinases at 10 nM (L) or 100 nM (H) concentrations or with enzyme storage buffer (-). (D) PIP 2 kinase assays with PI3Kα after incubation with subset of the GCK family, performed as described in (C). (E) PIP 2 kinase assays with the class I PI3K isoforms (PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ, 500 nM) after incubation with MST1.

Article Snippet: For phosphorylation reactions, predetermined concentrations of protein kinase and PI3K complexes were added to master mix containing 50 μM of ATP, 0.1 mg/mL of BSA and 1x Assay Buffer I (SignalChem) in 40 μl total volumes.

Techniques: Thin Layer Chromatography, Autoradiography, Produced, Recombinant, Incubation

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: PI3K Expression in microglia. (a) Immunocytochemistry images showing the expression of PI3K (red) in both BV2 microglial cell line (left) and rat primary microglia (right). Nuclei are stained blue with DAPI, and microglia are stained green with Lectin. (b) PI3K expression was also confirmed by western blotting. β‐Actin was used as an internal control. PI3K, phosphatidylinositol 3‐kinase [Color figure can be viewed at http://wileyonlinelibrary.com ]

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Expressing, Immunocytochemistry, Staining, Western Blot

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: HDAC inhibition upregulates PI3K/AKT pathway. (a) Western blot showing the upregulation of PI3K expression ( p = .0050), phosphorylation of AKT ( p = .0031) and CREB ( p = .0011), and also BDNF expression ( p = .0154) upon treatment with HDAC inhibitor (NaBu) in BV2 microglial cells, compared to control while the levels of total AKT and CREB remain unchanged. (b) Western blot data were quantified and normalized to β‐Actin. (c) Phospho‐AKT normalized to total AKT and phospho‐CREB normalized to total CREB. (d) Co‐treatment of HDAC inhibitor with PI3K inhibitor LY294002, confirmed by significant downregulation of pAKT ( p = .0005), resulted in a decrease in BDNF expression ( p = .0371). (e, f) Western blot data quantified and normalized to β‐Actin, phospho‐AKT normalized to total AKT. ( n = 3, * p < .05, ** p < .01, *** p < .001, compared with control). BDNF, brain‐derived neurotrophic factor; CREB, cAMP response element‐binding protein; PI3K, phosphatidylinositol 3‐kinase

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Inhibition, Western Blot, Expressing, Derivative Assay, Binding Assay

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: HDAC inhibition upregulates PI3K/AKT pathway. Immunocytochemistry images showing an increase in the expression of (a) PI3K (red), (b) phospho‐AKT (red), (c) phospho‐CREB, and (d) BDNF in rat primary microglia treated with HDAC inhibitor (NaBu) compared to that of control. Nuclei are stained blue with DAPI, and microglia are stained green with Lectin. Scale bar = 10 μm. BDNF, brain‐derived neurotrophic factor; CREB, cAMP response element‐binding protein; PI3K, phosphatidylinositol 3‐kinase [Color figure can be viewed at http://wileyonlinelibrary.com ]

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Inhibition, Immunocytochemistry, Expressing, Staining, Derivative Assay, Binding Assay

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: Enrichment of acetylation mark in Pik3ca. Chromatin immunoprecipitation (ChIP) assay shows an increase in H3K9ac enrichment in the Pik3ca promoter region upon 1 hr ( p = .0169) and 6 hr ( p = .01948) treatment with HDACi. Both time points show a significant increase in H3K9ac enrichment in the promoter of PI3K. H3K9ac enrichment was normalized against H3 enrichment to account for nucleosome density at the promoter regions probed. ( n = 4, * p < .05, ** p < .01, *** p < .001, compared with control). PI3K, phosphatidylinositol 3‐kinase

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Chromatin Immunoprecipitation

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: SUMO1 Knockdown downregulates PI3K/AKT pathway. (a) Western blot showing the downregulation of PI3K expression ( p = .0128) and phosphorylation of AKT ( p = .0007) in SUMO1 knockdown cells while the levels of total AKT remain unchanged. There was also decrease in the phosphorylation of CREB ( p = .0157) in SUMO1 knockdown cells while the levels of total CREB remain unchanged. The expression of BDNF ( p = .0209) was also downregulated in SUMO1 knockdown cells. SUMO1 knockdown also confirmed here by western blot ( p = .0005). (b) Western blot data were quantified and normalized to β‐Actin. (c) Phospho‐AKT normalized to total AKT and phospho‐CREB normalized to total CREB. (d) To restore PI3K function, active PI3K was added to the media leading to a significant increase in AKT phosphorylation ( p = .0024) followed by a rescue of BDNF induction ( p = .0056). (e, f) Western blot data quantified and normalized to β‐Actin, phospho‐AKT normalized to total AKT. ( n = 3, * p < .05, ** p < .01, *** p < .001, compared with control). BDNF, brain‐derived neurotrophic factor; CREB, cAMP response element‐binding protein; PI3K, phosphatidylinositol 3‐kinase

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Western Blot, Expressing, Derivative Assay, Binding Assay

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: SUMO1 knockdown downregulates PI3K/AKT pathway. Immunocytochemistry images showing a decrease in the expression of (a) PI3K (red), (b) pAKT (red), (c) pCREB (red), and (d) BDNF (red) in SUMO1 knockdown cells compared to control. Nuclei are stained blue with DAPI, and microglia are stained green with Lectin. Scale bar = 10 μm. BDNF, brain‐derived neurotrophic factor; CREB, cAMP response element‐binding protein; PI3K, phosphatidylinositol 3‐kinase [Color figure can be viewed at http://wileyonlinelibrary.com ]

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Immunocytochemistry, Expressing, Staining, Derivative Assay, Binding Assay

Journal: Glia

Article Title: Epigenetic regulation of microglial phosphatidylinositol 3‐kinase pathway involved in long‐term potentiation and synaptic plasticity in rats

doi: 10.1002/glia.23748

Figure Lengend Snippet: Whole‐cell patch‐clamp electrophysiological recordings showing the effect of microglial ablation on LTP. (a) Immunohistochemistry of hippocampal slices treated with clodronate shows a depletion of microglia in the hippocampal CA1 region. (b) Panel B depicts a hippocampal slice and the relative position of the recording (rec) and stimulating electrode (stim) in the stratum pyramidal (str. Pyr.) and stratum radiatum (str. Rad.), respectively. (c) LTP was induced in a hippocampal slice from a wil‐type rat by high‐frequency stimulation (HFS) combined with a depolarization to 0 mV 8 min after initial EPSC recording ( n = 5). The potentiation of the EPSC slope remained stable the whole period of the recording (50 min). (d) LTP was attenuated when the hippocampal slices were treated with clodronate (100 μg/mL) ( n = 6). (e) But when the slices were co‐treated with PI3K (1 μg/mL), LTP was restored in the ablated hippocampal slices ( n = 8). (f) Similarly, treatment with BDNF (200 ng/mL) could also ameliorate LTP in the clodronate‐treated slices. (g) The bar graph summarizes the average values of EPSC slope values during basal activity (−5 min) and 15 and 45 min post‐HFS, respectively. Asterisks represent significant difference in the EPSC potentiation between groups ( U test). Scale bars for EPSC traces are 50 pA and 40 ms. BDNF, brain‐derived neurotrophic factor; LTP, long‐term potentiation; PI3K, phosphatidylinositol 3‐kinase [Color figure can be viewed at http://wileyonlinelibrary.com ]

Article Snippet: Clodronate‐incubated hippocampal slices were then incubated with either active PI3K (1 μg/mL, Cat. No. P27‐18H‐10; SignalChem, USA) or BDNF (200 ng/mL, Cat. No. 3897; Cell Signalling Technologies, USA) 20 min before cells were patched.

Techniques: Patch Clamp, Immunohistochemistry, Activity Assay, Derivative Assay