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Proteome analysis of NGF stimulated PC12 cells

ABSTRACT: We used quantitative mass spectrometry-based proteomics to unravel global nerve growth factor (NGF)-induced changes in protein abundance using the rat PC12 cell line. Cells were stimulated with NGF for 0, 24 and 48 h in a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10). All experiments were performed as biological replicates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Adrenal Medulla Chromaffin Cell, Adrenal Medulla

DISEASE(S): Adrenal Gland Pheochromocytoma

SUBMITTER: Kristina Bennet Emdal

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD001478 | Pride | 2015-05-06

REPOSITORIES: Pride

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Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

Emdal Kristina B KB Pedersen Anna-Kathrine AK Bekker-Jensen Dorte B DB Tsafou Kalliopi P KP Horn Heiko H Lindner Sven S Schulte Johannes H JH Eggert Angelika A Jensen Lars J LJ Francavilla Chiara C Olsen Jesper V JV

Science signaling 20150428 374

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to ...[more]

PMID: 25921289

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Proteomics of NGF-TrkA signaling dynamics in neuroblastoma cells

Project description:We used quantitative mass spectrometry-based proteomics to unravel global nerve growth factor (NGF)-induced TrkA signaling dynamics at the interactome, phosphoproteome and proteome level. A tetracycline-inducible system for TrkA expression was generated in the human neuroblastoma cell line, SH-SY5Y. TrkA-induced cells were stimulated with NGF for different time points to follow phosphoproteome, interactome and proteome changes on a temporal scale. In a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10), the samples were stimulated with NGF as indicated. Phosphoproteome: 0, 10, 45 min and 0, 120, 120 min+cycloheximide. Interactome: 0, 5, 10 min. Proteome: 0, 24, 48 h. All experiments were performed as biological replicates.

2015-05-06 | PXD001115 | Pride

Proteomics of NGF-TrkA signaling dynamics in neuroblastoma cells

2017-03-31 | MSV000080834 | MassIVE

Human Sumoylome (SUMO-2) upon bacterial infection

Project description:Given the role of SUMOylation in pathogenic infection, we wanted to evaluate the changes to the host SUMO-2 subproteome during Shigella infection. HeLa cells overexpressing TAP-SUMO-2 or TAP-empty were used in conjunction with SILAC (Stable Isotope Labeling of Amino acids in Culture) (Golebiowski et al, 2009, 2010). Cells were grown in Dulbecco’s modified Eagle’s medium except that L-arginine and L-lysine were replaced with stable isotope (SILAC) forms depending on the treatment. Medium was supplemented with 10% dialyzed fetal calf serum. SILAC experiment compared TAP-containing cells (Lys0 and Arg0) with invasive Shigella flexneri (M90T) infected TAP-SUMO-2-containing cells (4,4,5,5-D4-lysine, Lys4, and 13C6- arginine, Arg6) and TAP-SUMO-2-containing cells infected with (mxiD) non-invasive strain of Shigella (13C6 15N2-lysine, Lys8, and 13C6 15N4 -arginine, Arg10).

2018-10-19 | PXD001100 | Pride

Proteomics analysis of synergistic drug combination of midostaurin and BMS-754807 in Ewing sarcoma ASP14 cell line

Project description:We used quantitative mass spectrometry-based proteomics to unravel global changes in the phosphoproteome upon treatment of ASP14 cells (a Ewing Sarcoma cell line) according to the setup below. The combination of drugs were experimentally shown to infer synergy in terms of cell killing. Three experiments were performed using a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10): Experiment 1: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: 0.1% DMSO Experiment 2: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: Midostaurin (90 nM) Heavy SILAC: BMS-754807 (20 nM) Experiment 3: Light SILAC: Drug combination (90 nM Midostaurin + 20 nM BMS-754807) Medium SILAC: BMS-754807 (20 nM) Heavy SILAC: 0.1% DMSO ASP14 cells were starved by replacing complete SILAC medium with SILAC minimal medium without serum over night. Cells were treated with drugs as stated above for 2h; then they were stimulated with serum for 20 minutes and harvested thereafter.

2017-04-19 | PXD002765 | Pride

Proteomics of late signaling responses decipher the roles of Cbl and Cbl-b in neuroblastoma cell differentiation

Project description:We applied quantitative mass spectrometry (MS)-based proteomics to study the roles of Cbl and Cbl-b in long-term signaling responses related to neurite outgrowth and differentiation of SH-SY5Y neuroblastoma cells. Using stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tag (TMT)-labeling in combination with off-line high-pH reversed-phase fractionation and LC-MS/MS we analyzed how Cbl and Cbl-b depletion by siRNA affected the proteome, phosphoproteome and ubiquitylome of the neuroblastoma cells. SILAC proteome SILAC (Light Arg0/Lys0, medium Arg6/Lys4, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 72 hours. For combined stimulation with ligand cocktail (FGF-2, IGF-1 PDGF-BB, TGFα) cells were treated with ligands for 48 h. Samples were analyzed in triplicates with set-up as described below: Set-up 1, 3 replicates (R1-3): Light: siGFP, Heavy: siCbl/siCbl-b Set-up 2, 3 replicates (E1-3): Light: siGFP + ligand cocktail, Medium: siCbl/siCbl-b, Heavy: siCbl/siCbl-b + ligand cocktail TMT phosphoproteome and proteome SH-SY5Y cells were treated with Cbl and Cbl-b siRNA, control (GFP) siRNA or Retinoic acid (RA) for 24 hours. Samples were prepared in triplicates and labelled with TMT10-plex reagents according to the set-up below: TMT10-126: siGFP E1 TMT10-127N: siCbl/siCbl-b E1 TMT10-127C: Retinoic acid E1 TMT10-128N: siGFP E2 TMT10-128C: siCbl/siCbl-b E2 TMT10-129N: Retinoic acid E2 TMT10-129C: siGFP E3 TMT10-130N: siCbl/siCbl-b E3 TMT10-130C: Retinoic acid E3 TMT10-131: Mix of the 9 samples SILAC Ubiquitin pulldown SILAC (Light Arg0/Lys0, heavy Arg10/Lys8) SH-SY5Y cells were treated with Cbl and Cbl-b or control (GFP) siRNA for 24 hours. Samples were analyzed in duplicates with set-up as described below: Light: siGFP, Heavy: siCbl/siCbl-b

2021-03-30 | PXD020389 | Pride

NGF Treatment of PC12 Cells

Project description:Expression profiling of PC12 cells stably transfected with GFP, GFP-SH2-Bb, or GFP-SH2-Bb(R555E), treated with or without NGF for 6h Keywords: response to NGF

2007-03-27 | GSE4557 | GEO

miRNA expression profile in PC12 cells under NGF treatment and deprivation

2013-05-11 | GSE46827 | GEO

miRNA expression profile in PC12 cells under NGF treatment and deprivation

Project description:miRNA profiling of PC12 rat pheochromocytoma cells during NGF-induced differentiation and apoptosis of differentiated cells after 24h of NGF deprivation. miRNA expression in differentiating and deprivated cells was compared to untreated and terminally differentiated PC12 cells respectivelly. Five-condition experiment, NGF-treated cells (1h, 3h, 6h, 24h, 240h) vs. untreated. One-condition experiment, NGF-deprivated vs. differentiated cells.

2013-05-11 | E-GEOD-46827 | biostudies-arrayexpress

MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

Project description:MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiae into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNA　microarray analysis using total RNAs harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis confirmed increased expression of miR-221, miR-181a* and miR-326, and decreased expression of miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Overexpression of miR-221 induced neurite outgrowth of PC12 cells in the absence of NGF treatment, and also enhanced neurite outgrowth caused by low-dose NGF. More importantly, knockdown of miR-221 by antagomir attenuated NGF-mediated neurite outgrowth. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are involved in apoptosis in PC12 cells. Our results indicate that miR-221 plays a critical role for neuronal differentiation as well as protection against apoptosis in PC12 cells.

2012-05-16 | GSE32122 | GEO

MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

Project description:MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiae into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNAM-cM-^@M-^@microarray analysis using total RNAs harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis confirmed increased expression of miR-221, miR-181a* and miR-326, and decreased expression of miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Overexpression of miR-221 induced neurite outgrowth of PC12 cells in the absence of NGF treatment, and also enhanced neurite outgrowth caused by low-dose NGF. More importantly, knockdown of miR-221 by antagomir attenuated NGF-mediated neurite outgrowth. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are involved in apoptosis in PC12 cells. Our results indicate that miR-221 plays a critical role for neuronal differentiation as well as protection against apoptosis in PC12 cells. NGF induced miRNAs expression in rat pheochromocytoma PC12 cells was measured in cells treated with 100 ng/ml NGF for 0, 12, 24 and 48 hr.

2012-05-15 | E-GEOD-32122 | biostudies-arrayexpress

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