Journal: The Journal of Biological Chemistry

Article Title: Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC 1 HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

doi: 10.1074/jbc.M109.043117

Figure Lengend Snippet: PACAP potently increases sympathetic Akt phosphorylation. A , primary sympathetic neuronal cultures were withdrawn from NGF (as in ) and treated with different concentrations of PACAP for 2 h before extraction and Western blot analyses. As with ERK, PACAP potently increased Akt phosphorylation. B , for studies with signaling pathway inhibitors, NGF-deprived cultures were pretreated with the indicated inhibitors for 15 min before peptide addition for 2 h. PI3K inhibitor LY294002 (25 μ m ) blocked PACAP-mediated Akt phosphorylation. MEK and PKC inhibitors, PD98059 (25 μ m ), and bisindolylmaleimide I ( BimI , 15 μ m ) and the inactive LY294002 analog LY303511 (25 μ m ) had little or no effects. Application of the PKA inhibitor H89 (25 μ m ) consistently potentiated PACAP-stimulated Akt phosphorylation. Data are representative of four independent experiments.

Article Snippet: For knockdown studies, 70% confluent cells in 24-well plates were transfected with siRNAs to mouse Gα q or PI3K p110γ subunit (Santa Cruz Biotechnology) using Lipofectamine 2000 (10 pmol of siRNA/2 μl of transfection reagent; Invitrogen).

Techniques: Phospho-proteomics, Extraction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC 1 HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

doi: 10.1074/jbc.M109.043117

Figure Lengend Snippet: Inhibition of Gα q and PI3Kγ signaling attenuates PACAP-stimulated Akt activation. A , sympathetic neuronal cultures were acutely withdrawn from NGF for 4 h to attenuate cellular Akt phosphorylation levels, then pretreated with 10 μ m AS252424 (15 min) to selectively inhibit PI3Kγ activity before 100 n m PACAP27 addition for 4 h. AS252424 blocked PACAP/PAC 1 receptor-stimulated Akt activation but did not attenuate NGF/TrkA-stimulated Akt phosphorylation (not shown). Identical results were obtained with PI3Kγ-selective inhibitor AS605240. B , treatment of stable AtT-20/PAC 1 HOP1 cultures with 100 n m PACAP27 for 4 h stimulated Akt phosphorylation; as in panel A with primary sympathetic neurons, the response was blocked upon inhibition with PI3Kγ-selective AS252424 (10 μ m ) treatment. AtT-20/PAC 1 HOP1 cultures were transfected at 70% confluency with mouse siRNA oligonucleotides to affect Gα q ( C ) or PI3Kγ ( D ) knockdown. After 48 h, the cultures were treated with 100 n m PACAP27 for 4 h. PACAP-stimulated Akt phosphorylation was attenuated when Gα q or PI3Kγ expression was diminished. Culture total Akt levels demonstrate loading equivalency across all samples. Representative data are from 2–4 experiments.

Article Snippet: For knockdown studies, 70% confluent cells in 24-well plates were transfected with siRNAs to mouse Gα q or PI3K p110γ subunit (Santa Cruz Biotechnology) using Lipofectamine 2000 (10 pmol of siRNA/2 μl of transfection reagent; Invitrogen).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Activity Assay, Transfection, Knockdown, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC 1 HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

doi: 10.1074/jbc.M109.043117

Figure Lengend Snippet: PACAP-mediated Akt signaling is neuroprotective in sympathetic neuronal cultures. A , shown is an experimental schematic for PACAP-mediated sympathetic neuronal survival. After NGF withdrawal (0 h) from immature sympathetic neuronal cultures, there is a 12–15-h survival window delaying neuronal commitment to apoptosis. If NGF is returned to the culture within the survival window ( a ), the cultures continue to live ( black solid arrows ) and do not undergo apoptosis programs. But if NGF is returned to the cultures outside of window parameters ( b ), the neurons are committed to apoptosis, and subsequent NGF addition is unable to affect rescue ( red broken arrows ). The addition of PACAP in place of NGF during the survival window extends the survival period ( c ) such that a much delayed addition of NGF ( blue bar ) can fully protect the neurons ( d ). Accordingly, the addition of signaling inhibitors ( hatched bars , e and f ) with PACAP during the window period will block the extended survival period and can be diagnostic of PACAP neurotrophic pathways. B , in this paradigm using the MTS survival assay ( lanes a–f correspond to those in panel A ), NGF replacement outside of the survival window did not offer neuroprotection when survival was assessed 48 h later ( b ). PACAP alone offered 50% protection ( c ) in NGF-deprived cultures, whereas the sequential addition of PACAP + NGF as described above was fully protective ( d ). Between ERK and Akt pathways, only LY294002 blocked the abilities for PACAP to promote neuronal survival in this experimental paradigm ( e ). As the MEK inhibitor PD98059 had no apparent effects ( f ), these results suggested that PI3K/Akt signaling may be more immediate in mediating PACAP survival signaling. Data represent the mean of 5–6 culture replicates ± S.E.; *, significantly different from control; +, different from PACAP-rescued cultures at p < 0.05.

Article Snippet: For knockdown studies, 70% confluent cells in 24-well plates were transfected with siRNAs to mouse Gα q or PI3K p110γ subunit (Santa Cruz Biotechnology) using Lipofectamine 2000 (10 pmol of siRNA/2 μl of transfection reagent; Invitrogen).

Techniques: Blocking Assay, Diagnostic Assay, Clonogenic Cell Survival Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC 1 HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

doi: 10.1074/jbc.M109.043117

Figure Lengend Snippet: Schematic of coordinate PACAP receptor signaling for sympathetic neuronal survival. The ability for the PAC 1 receptor to engage Gα s /Gα q signaling pathways represents a multifaceted but coordinate means for neurotrophic signaling. PAC 1 receptor Gα s signaling in sympathetic neurons can stimulate cAMP mechanisms to promote sustained ERK activation. In addition to rapid PAC 1 receptor-mediated Gα q activation of phospholipase C /PKC pathways, the internalization of PAC 1 receptors through clathrin-coated vesicles may facilitate scaffold assembly to engage PI3kγ/Akt signaling for survival mechanisms. Whether ERK, Akt, or other PAC 1 receptor-stimulated mechanisms attenuate JNK/p38 MAPK activation after NGF-deprivation remains to be elucidated. PDK , phosphoinositide-dependent protein kinase; AC , adenylyl cyclase.

Article Snippet: For knockdown studies, 70% confluent cells in 24-well plates were transfected with siRNAs to mouse Gα q or PI3K p110γ subunit (Santa Cruz Biotechnology) using Lipofectamine 2000 (10 pmol of siRNA/2 μl of transfection reagent; Invitrogen).

Techniques: Protein-Protein interactions, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Baicalein Potentiated M1 Macrophage Polarization in Cancer Through Targeting PI3Kγ/ NF-κB Signaling

doi: 10.3389/fphar.2021.743837

Figure Lengend Snippet: Baicalein is a potential inhibitor of PI3Kγ. (A) pharmacological network of baicalein (molecular ID: mol002714) and melanoma targets generated with Cytoscape V3.8.1. The property of yellow nodes are the common targets of drug and disease. All protein targets are represented by their gene symbols. (B) , protein-protein interaction (PPI) network of drug and disease target analysis. (C,D) the mRNA expression and protein levels of PI3Kγ p110 in THP-1-derived M1 macrophages with or without baicalein treatment. (E) the molecular docking of baicalein on PI3Kγ. (F) the binding curve of baicalein to PI3Kγ was measured by SPR. (G) the inhibitory effect of baicalein on PI3Kγ. Data are presented as mean ± SD and are obtained from at least independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, for comparison with control group. BA = baicalein.

Article Snippet: For the measurement of the kinase activity of PI3Kγ, ADP-Glo kinase assay kit (Promega, Madison, WI, United States, Cat#V910) and PI3Kγ (p110γ) assay kit (BPS Bioscience, San Diego, CA, Cat#79803) were used.

Techniques: Generated, Expressing, Derivative Assay, Binding Assay