Journal: Aging Cell

Article Title: Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs

doi: 10.1111/acel.12245

Figure Lengend Snippet: The effects of estrogen stimulation on the expression of miR-182, miR-223, and miR-142-3p and their targets in infant female quadriceps femoris -derived myoblasts. (A) Quantitative PCR analyses of miRNA transcripts normalized with RNU44 in human myoblasts treated for 72 h with 100 n m estradiol or mock. Note: The expression of miR-182 in these myoblasts was too low to be accurately measured. (B) qPCR analyses of target mRNAs (IGF-1, FOXO3A, FOXO1A) in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (C) Representative Western blots of target proteins, IGF-1R, FOXO3A and FOXO1A, and GAPDH, in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (D) Densitometry data of Western blots normalized with GAPDH. (E) Representative Western blots showing phosphorylation of AKT and mTOR proteins in myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (F) Densitometry data of Western blots normalized with GAPDH. Data are presented as percentage of control (mock) and reported as means ± SD of three independent experiments. OD indicates optical density. t -test, *** P

Article Snippet: Experiments for phosphorylation of AKT and mTOR Myoblasts were treated for 72 h with 10 nm E2 , 100 nm E2 , or solvent alone and analyzed in Western blots to detect phosphorylation of AKT (SER 473, Cell Signalling #9271) and mTOR (SER 2448, Cell Signalling #2971).

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature cell biology

Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

doi: 10.1038/s41556-019-0348-8

Figure Lengend Snippet: GSK-3 phosphorylates RacE at Ser192 in response to the chemoattractant. a , WT and RacE-KO cells were stimulated with the chemoattractant cAMP (1 μM). Total amounts of RacE and its phosphorylation at Ser192 were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). b , WT cells expressing GFP fused to WT RacE, phospho-defective RacE S192A or phospho-mimetic RacE S192D were stimulated with cAMP. Whole cell lysates prepared at the indicated time points were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(S192). c , The amino acid sequence in the vicinity of the phosphorylation site (Ser192, red) of RacE. A consensus motif for GSK-3 phosphorylation — a cluster of (S/TXXXS/T) — is underlined. A phosphopeptide used to raise anti-phospho-RacE (S192P) is highlighted. d , The location of serine 192 in a modeled RacE 3-D structure. (e–h) WT cells were treated with inhibitors to GSK-3 (250 nM LY2090314 in e and 10 mM lithium in f ), PI3K (250 μM LY294002 in g ), mTORC2 (0.5 μM PP242 in g ), or AKT (5 μM afuresertib in h ) for 10 min. Cells were then stimulated by the chemoattractant cAMP for the indicated amounts of time. Whole cell lysates were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). i , WT and cells lacking AKT (PkbA-KO or PkbR1-KO) were stimulated with cAMP for 30 s. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. j , Purified human GSK-3β was incubated with purified WT, GDP-bound RacE T25N or phospho-defective RacE S192A for 15 min. Ser192 phosphorylation of RacE was tested by immunoblotting. k , WT or GDP-bound RacE T25N was mixed with GSK-3β in the presence or absence of the GSK-3 inhibitor LY2090314 (10 nM) and examined for Ser192 phosphorylation using immunoblotting. l , Summary of the data. Experiments were repeated independently three times with similar results in a, b and e - k .

Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

Techniques: Expressing, Sequencing, Purification, Incubation

Journal: Nature cell biology

Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

doi: 10.1038/s41556-019-0348-8

Figure Lengend Snippet: RacE-GDP promotes chemoattractant-induced, mTORC2-mediated AKT phosphorylation in cells. The indicated Dictyostelium cell lines were stimulated with the chemoattractant cAMP (1 μM). a - f , WT cells and RacE-KO cells expressing different GFP-RacE constructs were analyzed. g and h , WT cells and RacE-KO cells expressing GFP-RacE were pretreated with 0.5 μM of the mTORC2 inhibitor PP242 for 10 min and then stimulated with cAMP. i and j , WT and RacE-KO cells expressing FLAG-tagged RasC or GTP-bound RasC Q62L were analyzed. a - j , Total amounts of two AKT homologs (PKBR1 and PKBA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls in a, c, e, g, and i . The band intensity of phosphorylated AKTs was quantified in b, d, f, h, and j : WT cells at 30 s (b, d, f and h) and WT cells expressing RasC at 30 s (j) were set at 100%. Values are average ± SD (n = 3 independent experiments).

Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

Techniques: Expressing, Construct, Activation Assay, Staining

Journal: Nature cell biology

Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

doi: 10.1038/s41556-019-0348-8

Figure Lengend Snippet: RacE-GDP specifically interacts with mTORC2. a , Dictyostelium cell lysates carrying GFP fused to the indicated forms of RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Quantification of interaction is shown. The band intensity of FLAG-Tor in immunoprecipitates of cells expressing WT RacE was set 100% (n=6, 6, 6 and 3 independent experiments for RacE, RacE T25N , RacE G20V and RacE T43A , respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between RacE and others. b , Dictyostelium cell lysates carrying the indicated GFP-RacE were subject to immunoprecipitation with GFP-Trap to analyze its association with endogenous PiaA. The band intensity of PiaA in immunoprecipitates of cells expressing WT RacE was set 100% (n = 3 independent experiments). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison betwen RacE with others. c , Dictyostelium cell lysates carrying GFP fused to the indicated forms of Rac1A and RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Experiment was repeated independently three times with similar results. d , HEK293T cells were transfected with YFP fused to the indicated constructs of human Rac1 and RhoA and subjected to immunoprecipitation using GFP-Trap. The band intensities of Tor and rictor in immunoprecipitates of cells expressing WT RhoA was set 100% (n = 3 and n = 4 independent experiments for Tor and Rictor, respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between YFP-RhoA and others. e , Summary of the data. f , AKTs are phosphorylated in the hydrophobic motif by mTORC2 and in the activation loop by PDK. g and h , WT, RacE-KO, and PiaA-KO Dictyostelium cells were stimulated with the chemoattractant cAMP (1 μM). (n = 3 independent experiments). The total amounts of two AKT homologs (PkbR1 and PkbA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls. The band intensity of phosphorylated AKTs was quantified in h : WT cells at 30 s were set at 100%. Values are average ± SD.

Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

Techniques: Incubation, Immunoprecipitation, Expressing, Transfection, Construct, Activation Assay, Staining

Journal: Nature cell biology

Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

doi: 10.1038/s41556-019-0348-8

Figure Lengend Snippet: Ser192 phosphorylated RacE-GDP forms a supercomplex with Tor and Ras-GTP. a and b , The indicated GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s. FLAG-RasC proteins were purified without cAMP stimulation. GFP-RacE was incubated with FLAG-RasC and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. c , GFP-RacE, GFP-RasC or GFP-RasG was incubated with FLAG-Tor that was purified in a high-salt condition and pulled down with GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. d , GFP fused to GDP-bound RacE T25N or GTP-bound RacE G20V were purified from Dictyostelium cells under a high salt condition after stimulation with the chemoattractant cAMP. These GFP fusion proteins were incubated with high-salt washed FLAG-Tor and/or FLAG-RasC proteins. GFP-RacE was pulled down with GFP-Trap, and the pellet fractions were analyzed by immunoblotting. e , RacE forms a complex with Tor and RasC. The indicated proteins were purified under high-salt conditions and mixed for 15 min at room temperature. GFP-RasC proteins were pulled down with GFP-Trap, and the pellet fraction was analyzed by immunoblotting. f and g , Different GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s in the presence or absence of the GSK-3 inhibitor LY2090314 (250 nM). GFP-RacE was incubated with FLAG-RasC in f or FLAG-Tor in g and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. h , Model for GPCR-mediated mTORC2-AKT signaling. In response to GPCR activation by chemoattractant, Rho-GDP becomes phosphorylated by GSK-3 and assembles the super signaling complex with Ras-GTP and mTORC2 to promote AKT phosphorylation. Experiments were repeated independently three times with similar results in a - g .

Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

Techniques: Purification, Incubation, Activation Assay

Journal: Nature cell biology

Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

doi: 10.1038/s41556-019-0348-8

Figure Lengend Snippet: Phosphorylated RacE-GDP activates mTORC2 in vitro . mTORC2-mediated AKT phosphorylation was reconstituted using purified proteins. mTORC2 (FLAG-Tor, -PiaA or -Lst8), FLAG-RacE, and FLAG-RasC/G were purified from Dictyostelium cells. +cAMP indicates that cells were stimulated by the chemoattractant for 30 s before purification of FLAG-RacE. Purified proteins were mixed in the presence or absence of ATP and human unactive AKT for 5 min at room temperature. AKT phosphorylation was analyzed by immunoblotting using anti-phospho AKT (serine 473) antibodies. a , mTORC2 phosphorylates AKT in the presence of RacE and RasC. b and c , mTORC2 activation requires PiaA and Lst8, but not the mSIN1 homolog Rip3. FLAG-PiaA or FLAG-Lst8 was added to FLAG-Tor purified from the indicated KO cell lines in b . FLAG-PiaA and/or FLAG-Lst8 were incubated with high-salt washed FLAG-Tor in c . d , mTORC2 activation requires RacE-GDP. Purified RacE was incubated with EDTA (25 mM), GTPγS (0.5 mM) or GTPγS then GDP (2.5 mM) (GTPγS → GDP) before reconstitution. e , WT RacE or GDP-bound RacE T25N , but not GTP-bound RacE G20V , activates mTORC2 after the chemoattractant stimulation. f , mTORC2 activation needs RasC-GTP but not RasG-GTP. g , RacE phosphorylation controls mTORC2 activation. Phospho-mimetic mutation S192D in GDP-bound RacE T25N activates mTORC2 without chemoattractant stimulation, while the phospho-defective S192A mutation blocks it. h , mTORC2 activation requires RasC-GTP. Purified RacC was incubated with EDTA followed by either GTPγS or GDP before reconstitution. i , Summary of the data. j , RacE G23V -GDP activates mTORC2. Purified RacE T25N and RacE G23V were incubated with EDTA followed by GTPγS or GDP prior to reconstitution. Experiments were repeated independently three times with similar results in a - h and j .

Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

Techniques: In Vitro, Purification, Activation Assay, Incubation, Mutagenesis

Journal: Redox Biology

Article Title: Melatonin protects mouse granulosa cells against oxidative damage by inhibiting FOXO1-mediated autophagy: Implication of an antioxidation-independent mechanism

doi: 10.1016/j.redox.2018.07.004

Figure Lengend Snippet: Inhibition of FOXO1 transcriptional activity through the melatonin-PI3K-AKT axis protects GCs from H 2 O 2 -induced autophagic PCD. (A) GCs transfected with Foxo1 siRNA or scrambled control siRNA for 24 h were cultured in media containing various concentrations of melatonin (0, 5, 10, 20 μM). 24 h later, cells were rinsed with PBS, and exposed to H 2 O 2 (200 μM) incubation for another 2 h. The expression of phosphorylated FOXO1 (p-FOXO1) was determined by western blotting. (B) The phosphorylation level of FOXO1 was quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. ** Represents P

Article Snippet: Antibodies against AKT (9272), phospho-AKT (4060), FOXO1 (2880), phospho-FOXO1 (9461), FLAG (2908), BECN1 (3495), MTOR (2983), ATG3 (3415), ATG5 (8540), ATG7 (2631), and ATG12 (4180) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Activity Assay, Transfection, Cell Culture, Incubation, Expressing, Western Blot

Journal: Nature Communications

Article Title: Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

doi: 10.1038/s41467-018-06136-x

Figure Lengend Snippet: PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c , d , the western blot experiment was repeated independently for three times with similar results

Article Snippet: FAK (#3285), phospho-FAK (Tyr397, #8556), Akt (#4685) and phospho-Akt (Ser473, #4060) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Variant Assay, Modification, Transfection, Plasmid Preparation, Molecular Weight, Western Blot

Journal: Frontiers in Behavioral Neuroscience

Article Title: Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

doi: 10.3389/fnbeh.2014.00455

Figure Lengend Snippet: Total sniffing time (means ± SEM sec) of WT, Akt1 +/− , Nrg1 +/− , and double mutant mice in the sociability and social recognition task of Experiment 2.2. (A) In the 5-min sociability test, all mice (except double mutant mice) displayed a significant social preference toward the stimulus (i.e., stranger 1) mouse. (B) In the 5-min social recognition test (5-min after the end of the sociability test), only WT mice displayed a significant preference toward a novel (i.e., stranger 2) mouse. * p

Article Snippet: The expression levels of Akt1 and Nrg1 proteins in the cerebral cortex of adult mice (n = 5/group, the second cohort) were measured using Western blot with Akt1 (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA), Gapdh (1:5000; Cell Signaling Technology), and Nrg1 (1:1000; Santa Cruz Biotechnology, Inc., CA, USA) antibodies.

Techniques: Size-exclusion Chromatography, Mutagenesis, Mouse Assay

Journal: Frontiers in Behavioral Neuroscience

Article Title: Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

doi: 10.3389/fnbeh.2014.00455

Figure Lengend Snippet: General features of wild-type (WT), Akt1 +/− , Nrg1 +/− , and double mutant mice in Experiment 1. (A) Physical growth at postnatal day (PD) 30, 60, and 197. (B) Expression of Akt1 protein levels in the cerebral cortices of the 4 groups of mice. A ~30–40% reduction of Akt1 protein was found in both Akt1 +/− and double mutant mice. (C) The expression of Nrg1 protein level in the cerebral cortices of the 4 groups of mice. A ~40–45% reduction of Nrg1 protein was found in both Nrg1 +/− and double mutant mice. (D) Representative PET-CT fusion images of coronal slices of the 4 groups of mice using microPET with 18 F-FDG. The medial prefrontal cortices (mPFC, Bregma 1.96 mm) are highlighted in blue rectangles, and the striatums (Bregma 0.5 mm) are highlighted in red polygons. (E) The normalized and averaged standardized uptake values (SUV) in the brain regions of interest (ROI, including the mPFC and striatum) among the 4 groups. The ratio was calculated as SUV target ROI (FDG) /SUV cerebellum (FDG) . * p

Article Snippet: The expression levels of Akt1 and Nrg1 proteins in the cerebral cortex of adult mice (n = 5/group, the second cohort) were measured using Western blot with Akt1 (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA), Gapdh (1:5000; Cell Signaling Technology), and Nrg1 (1:1000; Santa Cruz Biotechnology, Inc., CA, USA) antibodies.

Techniques: Mutagenesis, Mouse Assay, Expressing, Positron Emission Tomography

Journal: Frontiers in Behavioral Neuroscience

Article Title: Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

doi: 10.3389/fnbeh.2014.00455

Figure Lengend Snippet: Total sniffing time and number of calls (means ± SEM) of WT, Akt1 +/− , Nrg1 +/− , and double mutant mice in the social interaction and social communication task in Experiment 3. (A) The total anogenital sniffing time during a 5-min direct encounter with an estrous female mouse. (B,C) The total sniffing time during a 5-min non-stimulus baseline and a 5-min indirect social encounter with another male or an ovariectomized (OVX) female, respectively. (D,E,F) Total number of ultra-sonic vocalization (USV) calls during a 5-min non-stimulus baseline and a 5-min indirect social encounter with another male, an OVX female, and 15 µl of fresh female urine, respectively. * p

Article Snippet: The expression levels of Akt1 and Nrg1 proteins in the cerebral cortex of adult mice (n = 5/group, the second cohort) were measured using Western blot with Akt1 (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA), Gapdh (1:5000; Cell Signaling Technology), and Nrg1 (1:1000; Santa Cruz Biotechnology, Inc., CA, USA) antibodies.

Techniques: Mutagenesis, Mouse Assay

Journal: Cell Cycle

Article Title: Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

doi: 10.1080/15384101.2018.1562286

Figure Lengend Snippet: Decreased PDYN expression and suppressed PI3K/Akt/Nrf2/HO-1 pathway in epileptiform hippocampal neurons MDA content and SOD activity (a) using commercial kits, cell apoptosis (b) using flow cytometry, mRNA expression of DYN and HO-1 (c) using RT-qPCR, and protein expression of PDYN, total Nrf2, nuclear Nrf2, HO-1, PI3K, as well as phosphorylation level of Akt (p-Akt) (d-e) using western blot, in cultured hippocampal neurons exposed to Mg 2+ -free solution or not. *p

Article Snippet: After being blocked with 5% non-fat milk, the membrane was incubated with the following primary antibodies: anti-PDYN (OriGene, Rockville, MD, USA), anti-PI3K (Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Akt (Santa Cruz Biotechnology), and anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Beyotime).

Techniques: Expressing, Multiple Displacement Amplification, Activity Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Cell Cycle

Article Title: Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

doi: 10.1080/15384101.2018.1562286

Figure Lengend Snippet: Decreased PDYN expression and suppressed PI3K/Akt/Nrf2/HO-1 pathway in a rat model of epilepsy Serum levels of TNF-α, IL-2, and IL-6 (a) using ELISA, MDA content and SOD activity (b) using commercial kits in control and epileptic model rats. In situ cell apoptosis (c) using TUNEL staining (Scale bar: 25.0 μm in overview; 2 μm in CA1 and CA3), mRNA expression of PDYN and HO-1 (d) using RT-qPCR, and protein expression of PDYN, total Nrf2, nuclear Nrf2, HO-1, PI3K, as well as phosphorylation level of Akt (p-Akt) (e) using western blot, in the hippocampus of control and epileptic model rats. n = 8/group. *p

Article Snippet: After being blocked with 5% non-fat milk, the membrane was incubated with the following primary antibodies: anti-PDYN (OriGene, Rockville, MD, USA), anti-PI3K (Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Akt (Santa Cruz Biotechnology), and anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Beyotime).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Activity Assay, In Situ, TUNEL Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Cell Cycle

Article Title: Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

doi: 10.1080/15384101.2018.1562286

Figure Lengend Snippet: Dynorphin activation of KOR alleviated seizure-like neuron injury via activation of PI3K/Akt/Nrf2/HO-1 pathway MDA content and SOD activity (a) using commercial kits, cell apoptosis (b) using flow cytometry, mRNA expression of HO-1 (c) using RT-qPCR, and protein expression of total Nrf2, nuclear Nrf2 and HO-1 (d) using western blot, in cultured hippocampal neurons exposed to Mg 2+ -free solution, followed by treatment with dynorphin-A and LY294002 (an inhibitor of PI3K/Akt pathway)/ZnPPIX (an HO-1 inhibitor), both alone or in combination. Cultured hippocampal neurons without any treatment served as the control group. DMSO served as the vehicle of LY294002 and ZnPPIX. Dyn-A, dynorphin-A. LY, LY294002. *p

Article Snippet: After being blocked with 5% non-fat milk, the membrane was incubated with the following primary antibodies: anti-PDYN (OriGene, Rockville, MD, USA), anti-PI3K (Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Akt (Santa Cruz Biotechnology), and anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Beyotime).

Techniques: Activation Assay, Multiple Displacement Amplification, Activity Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Cell Cycle

Article Title: Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

doi: 10.1080/15384101.2018.1562286

Figure Lengend Snippet: Dynorphin activation of KOR activated PI3K/Akt/Nrf2/HO-1 pathway and alleviated seizure-like neuron injury MDA content and SOD activity (a) using commercial kits, cell apoptosis (b) using flow cytometry, mRNA expression of HO-1 (c) using RT-qPCR, and protein expression of total Nrf2, nuclear Nrf2, HO-1, PI3K, as well as phosphorylation level of Akt (p-Akt) (d) using western blot, in cultured hippocampal neurons exposed to Mg 2+ -free solution, followed by treatment with the KOR agonist dynorphin-A and the KOR antagonist GNTI, both alone or in combination. Cultured hippocampal neurons without any treatment served as the control group. Dyn-A, dynorphin-A. *p

Article Snippet: After being blocked with 5% non-fat milk, the membrane was incubated with the following primary antibodies: anti-PDYN (OriGene, Rockville, MD, USA), anti-PI3K (Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Akt (Santa Cruz Biotechnology), and anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Beyotime).

Techniques: Activation Assay, Multiple Displacement Amplification, Activity Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Cell Cycle

Article Title: Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

doi: 10.1080/15384101.2018.1562286

Figure Lengend Snippet: PDYN overexpression alleviated pilocarpine-induced epilepsy and neuronal apoptosis in rats Serum levels of TNF-α, IL-2, and IL-6 (a) using ELISA, MDA content and SOD activity (b) using commercial kits, in the groups of model, LV-NC, and LV-Dyn. In situ cell apoptosis (c) using TUNEL staining (scale bar: 25.0 μm in overview; 2 μm in CA1 and CA3), mRNA expression of PDYN (d) and HO-1 (e) using RT-qPCR, and protein expression of PDYN, total Nrf2, nuclear Nrf2, HO-1, PI3K, as well as phosphorylation level of Akt (p-Akt) (f) using western blot, in the rat hippocampus in the groups of model, LV-NC, and LV-PDYN. n = 8/group. *p

Article Snippet: After being blocked with 5% non-fat milk, the membrane was incubated with the following primary antibodies: anti-PDYN (OriGene, Rockville, MD, USA), anti-PI3K (Cell Signaling Technology Inc., Danvers, MA, USA), anti-p-Akt (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Akt (Santa Cruz Biotechnology), and anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Beyotime).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Activity Assay, In Situ, TUNEL Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: The motion curves of rats in the spatial probe test (SPT) of rats in the five groups Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E, PI3K/Akt agonist group.

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Single-particle Tracking

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: Histopathologic changes in the hippocampal tissue detected by HE staining Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E, PI3K/Akt agonist group.

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Staining

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: Expression of PI3K-AKT-mTOR pathway proteins in the cerebral cortex A. , hippocampus B. , cerebellum C. , brain stem D. , and thalamus E. of rats among the five groups. Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E, PI3K/Akt agonist group.

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: The proportion of δ waves in electrocorticogram (ECoG) of rats in the five groups before and after drug administration Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E: PI3K/Akt agonist group. A: 5 min before drug administration; B: 5 min after drug administration; The superscript lowercase letters ( a, b, c, d, e ) represent the pairwise comparison among the five groups with the same protein and tissue. The same superscript lowercase letter indicated the P > 0.05, and the different superscript lowercase letter indicated the P

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques:

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: The comparisons of the duration of loss of righting reflex (LORR) and ataxic period of anesthetized rats among the three groups Note: Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; A: comparison of the duration of LORR; B: comparison of the ataxic period; # , compared with Group B, P

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques:

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: Graph of the average escape latency (EL) of rats in the place navigation test Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E, PI3K/Akt agonist group.

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques:

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: The mRNA expression in the PI3K-AKT-mTOR pathway in different tissues Note: Group A, blank control group; Group B, anesthetized rat model group; Group C, anesthetized rat model + PI3K/Akt agonist group; Group D, anesthetized rat model + PI3K/Akt antagonists group; Group E, PI3K/Akt agonist group. The comparison of mRNA expression of PI3K A. , AKT B. , mTOR C. and P70S6K D. in cerebral cortex, hippocampus, cerebellum, brain stem, and thalamus of rats among the five groups. The superscript lowercase letters ( a, b, c, d ) represent the pairwise comparison among the five groups with the same protein and tissue. The same superscript lowercase letter indicated the P > 0.05, and the different superscript lowercase letter indicated the P

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: The PI3K-AKT-mTOR pathway activates recovery from general anesthesia

doi: 10.18632/oncotarget.10172

Figure Lengend Snippet: The dynamic changes of electrocorticogram (ECoG) in rats before and after drug administration Note: Group A. , blank control group; Group B. , anesthetized rat model group; Group C. , anesthetized rat model + PI3K/Akt agonist group; Group D. , anesthetized rat model + PI3K/Akt antagonists group; Group E . PI3K/Akt agonists group A: ECoG image of rats in Group A; B1-B2: ECoG image of rats in Group B at the time points of 5 min before (B1) and after (B2) drug administration; C1-C2: ECoG image of rats in Group C at the time points of 5 min before (C1) and after (C2) drug administration; D1-D2: ECoG image of rats in Group D at the time points of 5 min before (D1) and after (D2) drug administration; E1-E2: ECoG image of rats in Group E at the time point of 5 min before (E1) and after (E2) drug administration.

Article Snippet: Goat anti-rat monoclonal antibodies for PI3K, AKT, p-AKT, mTOR, p-mTOR, P70S6K and p-P70S6K were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques:

Journal: Neoplasia (New York, N.Y.)

Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

doi: 10.1016/j.neo.2018.10.009

Figure Lengend Snippet: SDS22 regulates AKT and MAPK pathway at physiological level to inhibit malignancy. (A) Whole cell lysates of MCF7 cells stably expressing either scramble (NS) or unrelated SDS22 shRNAs were immunoblotted for the indicated proteins. MCF7 cells were stably knocked down using three unrelated shRNAs against SDS22 through lentivirus transduction. (B) Soft agar colony formation assay of MCF7 cells expressing either scramble (NS) or SDS22 shRNAs in the absence and presence of AKT/MEK inhibitor. (C) Scratch wound healing assay of MCF7 cells stably expressing either NS or SDS22 shRNA. Cells were grown in the presence or absence of AKT/MEK inhibitor as indicated. (D) Quantification of cell migration from scratch wound healing data of panel (C). Cell migration of NS cells at 24 hours was taken as 100%. (E) Cell invasion of MCF7 cells stably expressing either NS or SDS22 shRNA by Boyden chamber assay. Cells were grown in the presence or absence of AKT/MEK inhibitor as indicated. (F) Quantification of cell invasion from data of panel E. Cell invasion of NS cells was taken as 100%. ** P

Article Snippet: Antibodies against the SDS22, GFP, PARP1, phospho AKT(S473), phospho AKT(T308), AKT, PARP1, MEK, ERK, p-cJUN, Twist, Snail, p53, APAF-1, Bcl2, BAX ATM, and AKT were obtained from Santa Cruz Biotechnology (Santacruz, CA).

Techniques: Stable Transfection, Expressing, Transduction, Soft Agar Assay, Wound Healing Assay, shRNA, Migration, Boyden Chamber Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

doi: 10.1016/j.neo.2018.10.009

Figure Lengend Snippet: SDS22 inactivates AKT and MAPK–ERK signaling pathway through their dephosphorylation. (A) Whole cell lysates of MDA-MB-231 cells ectopically expressing either vector control or SDS22 for 48 hours were immunoblotted for the indicated proteins. (B) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells for the indicated proteins. Whole cell lysates were immunoprecipitated with either the IgG control or SDS22 antibody. (C) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells. Whole cell lysates were immunoprecipitated with either the IgG control or AKT/MEK/ERK antibody. Ratio of SDS22 and ERK/MEK/AKT was quantified by the Image J software. (D) Immunoblotting of whole cell lysates of MCF7 cells expressing either the vector control or wild-type SDS22 or mutants SDS22 for 48 hours. (E) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells ectopically expressing the indicated plasmids for 48 hours. Whole cell lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins.

Article Snippet: Antibodies against the SDS22, GFP, PARP1, phospho AKT(S473), phospho AKT(T308), AKT, PARP1, MEK, ERK, p-cJUN, Twist, Snail, p53, APAF-1, Bcl2, BAX ATM, and AKT were obtained from Santa Cruz Biotechnology (Santacruz, CA).

Techniques: De-Phosphorylation Assay, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Immunoprecipitation, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

doi: 10.1016/j.neo.2018.10.009

Figure Lengend Snippet: SDS22 inhibits EMT process by limiting the activity of AKT kinase. (A) Phase contrast image of morphology of MCF7 cells stable expressing either NS or SDS22 shRNA. (B) Whole cell lysates of MCF7 cells expressing either NS or SDS22 shRNA were immunoblotted for the indicated proteins. SDS22 knockdown cells were grown in the absence or presence of 5 μM AKT inhibitor for 12 hours. (C) Real-time RT-PCR demonstrated the relative mRNA levels of EMT regulators. Expression levels of regulators in NS cells were normalized with loading control GAPDH and taken as 1. (D) Whole cell lysates of MDA-MB-231 cells expressing either vector or SDS22 were immunoblotted for the indicated proteins. (E) Immunofluorescence staining was performed to detect the expression levels of E-cadherin. Red color represents E-cadherin, and blue color represents DAPI staining. (F) Quantification of expression levels of E-cadherin following overexpression of SDS22 as in panel E.* P

Article Snippet: Antibodies against the SDS22, GFP, PARP1, phospho AKT(S473), phospho AKT(T308), AKT, PARP1, MEK, ERK, p-cJUN, Twist, Snail, p53, APAF-1, Bcl2, BAX ATM, and AKT were obtained from Santa Cruz Biotechnology (Santacruz, CA).

Techniques: Activity Assay, Expressing, shRNA, Quantitative RT-PCR, Multiple Displacement Amplification, Plasmid Preparation, Immunofluorescence, Staining, Over Expression

Journal: Neoplasia (New York, N.Y.)

Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

doi: 10.1016/j.neo.2018.10.009

Figure Lengend Snippet: SDS22 expression is conversely correlated with active form of AKT in breast cancer patient samples. (A) Immunohistochemical analysis of SDS22, pAKT473, and pAKT308 in different grades of human breast cancer samples. (B) Statistical analysis of the average score of SDS22 and active form of AKT staining between cancer tissues and corresponding nontumor tissues, P

Article Snippet: Antibodies against the SDS22, GFP, PARP1, phospho AKT(S473), phospho AKT(T308), AKT, PARP1, MEK, ERK, p-cJUN, Twist, Snail, p53, APAF-1, Bcl2, BAX ATM, and AKT were obtained from Santa Cruz Biotechnology (Santacruz, CA).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages

doi: 10.3389/fimmu.2018.01419

Figure Lengend Snippet: GPR84 signaling activates AKT, ERK, and NFκβ in WT, but not in GPR84 −/− macrophages. (A) Bone marrow-derived macrophages (BMDMs) were treated with 0.1 µg/ml LPS for 2 h before stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 1, 5, 10, 30, and 60 min. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments are shown. (B) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with vehicle (0.3% DMSO) or 1 µM 6-OAU for 30 min followed by p65 staining. Confocal microscopy images are illustrative of two separate experiments. (C) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 5, 10, 30, and 60 min. Western blotting for p65 was performed using samples from cytoplasmic and nuclear fractions of cell lysates, followed by stripping and re-staining for histone-3 in the nuclear fraction and α-tubulin in the cytoplasmic fraction as a loading control. Representative images from n = 3 independent experiments are shown.

Article Snippet: Rabbit anti-phospho-ERK (D13.14.4E), rabbit anti-total-ERK, rabbit anti-phospho-Akt (D9E), rabbit anti-total-Akt, β-actin, were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Derivative Assay, Western Blot, Stripping Membranes, Staining, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages

doi: 10.3389/fimmu.2018.01419

Figure Lengend Snippet: GPR84 antagonist abrogates the enhanced inflammatory response mediated by 6-OAU. (A) Intracellular cyclic AMP levels were measured in CHO-GPR84 cells pre-treated with the antagonist followed by forskolin and 6-OAU stimulation. Data are represented as the mean ± SEM of the percentage of the response to forskolin in the absence of agonists n = 3 independent experiments. (B–H) Bone marrow-derived macrophages were treated with LPS (0.1 µg/ml) for 2 h before pre-treatment with vehicle (0.3% DMSO), 10 µM antagonist for 30 min, or 200 ng/ml Pertussis toxin for 90 min. Afterward cells were stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for either 10′ for protein isolation or 1 h for RNA extraction. (B) Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments. (C–H) mRNA expression of Tnf α (C) , Il-6 (D) , Il-12 (E) , Ccl5 (F) , Ccl2 (G) , and Cxcl1 (H) was analyzed by q-PCR. Data are presented as mean ± SEM of n = 4–7 separate experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison post hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 versus vehicle; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 versus 6-OAU.

Article Snippet: Rabbit anti-phospho-ERK (D13.14.4E), rabbit anti-total-ERK, rabbit anti-phospho-Akt (D9E), rabbit anti-total-Akt, β-actin, were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Techniques: Derivative Assay, Isolation, RNA Extraction, Western Blot, Stripping Membranes, Staining, Expressing, Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Pegylated liposomal-paclitaxel induces ovarian cancer cell apoptosis via TNF-induced ERK/AKT signaling pathway

doi: 10.3892/mmr.2018.8811

Figure Lengend Snippet: ERK/AKT signaling pathway is involved in the activation of the TNF/caspase-3 cascade within ovarian cancer cells. (A) Inhibition of ERKIR decreased caspase-3 expression levels within CAOV-3 cells compared with in the control group. (B) Inhibition of ERKIR inhibited TNF-induced CAOV-3 cell apoptosis compared with in the control group. (C) PL-PTX-induced CAOV-3 cell apoptosis was inhibited via an ERK inhibitor compared with in the control group. **P

Article Snippet: The primary antibodies used were against: Caspase-9 (1:1,200; ab32539), ERK (1:1,000; ab54230) and phosphorylayed (p)ERK 1/2 (phospho-Thr202/Tyr204; 1:1,000; ab214362), caspase-3 (1:1,200; ab2171), protein kinase B (AKT; 1:1,000; ab8805), p-AKT (phosphor-S473; 1:1,000; ab8932) and β-actin (1:500; ab8226; all Abcam) for 12 h at 4°C.

Techniques: Activation Assay, Inhibition, Expressing