MRNA Profiling of Polysome fractions from #483 T-ALL treated with 4EGI-1 or DMSO
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Adrian Schwarzer
PROVIDER: E-GEOD-41382 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 M-NM-<M 4EGI-1 or DMSO. After 2 h incubation at 37M-BM-0C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 M-bM-^@M-^S 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 M-BM-0C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 M-BM-0C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturerM-BM-4s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 M-BM-5l of RNase-free water. RNA concentrations were determined and the samples were stored at -80 M-BM-0C. The cytosolic/total- RNA sample for normalization was obtained from the same samples (106 treated/non-treated cells) using the standard Trizol protocol.
2015-02-01 | E-GEOD-56150 | biostudies-arrayexpress
Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
2019-10-15 | GSE134102 | GEO
Project description:DO11.10 x TCR-Calpha-/- T cells stimulated under Th2 conditions for 6 days were centrifuged over Histopaque 1083 (Sigma) to remove dead cells, washed and rested for 20 min at 37C in complete media with 0.1 mg/ml cycloheximide (CHX, Sigma) added for the final 10 min. Restimulated cells were activated with 10 ug/ml plate-bound I-Ad/ova complexes for 3 hrs. Cells were washed with ice-cold PBS/CHX and resuspended in polysome extraction buffer [30 mM HEPES (pH 7.4), 15 mM MgCl2, 0.3 M NaCl, 1% Triton X-100, 0.1 mg/ml CHX, 1 mg/ml heparin, 500 U/ml SUPERase-In RNAse inhibitors (Ambion, Austin, TX), 1x Complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN)]. After incubation on ice for 25 min, nuclei and debris were removed by centrifuging and the cleared supernatants transferred to fresh tubes. 1.5 ml of each extract (from 3 x 10exp8 T cells) were layered on a 37.5 ml 10-50% sucrose gradient prepared in SW28 ultraclear centrifuge tubes (Beckman Coulter, Fullerton, CA) using the salts described above, but without detergent, RNAsin or protease inhibitors. After centrifugation for 220 min at 28,000 rpm (average RCF = 103,745), fractions were isolated using an ISCO density gradient fractionator (Lincoln, NE) by pumping 60% sucrose into the bottom of the tube and collecting the displaced volumes into acid phenol:chloroform, with constant monitoring of ultraviolet absorbance at 254 nm. Extracted RNA was precipitated with isopropanol, washed and resuspended in distilled water. RNA from polysome associated fractions 7-12 of the gradients described above were pooled and 30-100 ug of total RNA per sample were used to template a Superscript III reverse transcription reaction (Invitrogen, Carlsbad, CA) incorporating amino-allyl dUTP (Sigma). cDNA samples were washed with distilled water on microcon-30 columns (Millipore, Billerica, MA), dried and labeled with Cy3 (primed samples) or Cy5 (restimulated samples) using CyDye reactive dye labeling kits (Amersham). Unincorporated dye was removed using QiaQuick PCR purification columns (Qiagen), labeled cDNAs were eluted in 10mM Tris, pH 8, combined and dried. Fluorescence in the Cy3 and Cy5 channels was acquired using an Axon 4000B microarray scanner and GenePix software (Axon Instruments, Union City, CA). Data were normalized using the R package Bioconductor software and lowess normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996). Keywords = Th2 differentiation Keywords = translation Keywords = polysome profile Keywords: cell stimulation
2006-03-23 | GSE4500 | GEO
Project description:The aim of the project was to purify Xenopus replisome from replicationg chromatin assembled in Xenopus laevis egg extract. To this end recombinant Cdc45-TEV-His10-FLAG5 was expressed in bacteria and purified. 4 ml of Xenopus laevis egg extract was activated into interphase and supplemented with 10 ng/µl of demembranated sperm DNA, 70 nM recombinant Cdc45, 40 µM aphidicolin, 5 mM caffeine and incubated at 23°C for 60 min. Chromatin was isolated in ANIB100 buffer (50 mM HEPES pH 7.6, 100 mM KOAc, 10 mM MgOAc, 2.5 mM Mg-ATP, 0.5 mM spermidine, 0.3 mM spermine, 1 µg/ml of each aprotinin, leupeptin and pepstatin, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 and 10 mM 2-chloroacetamide) as described previously (Gillespie, Gambus et al. 2012). Chromatin pellets re-suspended in ANIB100 containing 20% sucrose. Protein complexes were released from chromatin by digestion with 4 U/µl benzonase nuclease (E1014-25KU, Sigma) and sonicated for 5 min using a sonicator with settings: 30 sec on, 30 sec off, low power (Bioruptor Diagenode UCD-200). Immunoprecipitation was performed using 100 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich). Before elution the sample was washed four times with 1 ml of ANIB100 20% sucrose, ANIB100 20% sucrose 0.1% Triton X-100, ANIB100 and elution buffer (25 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgAc, 1 mM ATP and 0.02% NP-40), respectively. The sample was eluted adding 250 µM 3xFLAG peptide (Stratech) to 200 µl of elution buffer and a small proportion of it analysed by mass spectrometry.
2023-10-27 | PXD044422 | Pride
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:This series represents the data set from the paper <u>Assembly of microarrays for genome-wide measurement of DNA copy number</u> appearing in Nat Genet 2001 Nov;29(3):263-4 and it's web supplement. Specimen procurement, labeling, hybridization and imaging protocols from Nat Genet 2001 Nov;29(3):263-4 web supplement: <b>Specimens.</b> We obtained GM03563, GM00143, GM05296, GM07408, GM01750, GM03134, GM13330, GM03576, GM01535, GM07081, GM02948, GM04435, GM10315, GM13031 and GM01524 cell strains from the NIGMS Human Genetics Cell Repository (Coriell Institute for Medical Research), and cultured them under the recommended conditions. We cultured cell lines BT474, HCT116, T47D, MPE600, COLO320, SW837, MDA-MB-231, MDA-MB-453 and HT29 under the recommended conditions. To isolate DNA from confluent cell cultures, we collected cells from T75 flasks and then incubated them overnight at 55 C in a 3 ml solution containing 0.01 M Tris, pH 7.5, 0.001 M EDTA, pH 8, 0.5% SDS and 0.1 g/l proteinase K. We added 1 ml of saturated NaCl solution and 10 ml of ethanol to the cell lysate and collected the DNA by spooling. We removed excess liquid and then dissolved the DNA in 1 ml of H2O. We isolated DNA from breast tumor specimens as described previously. <b>DNA labeling.</b> We labeled DNA by nick translation or random priming. For nick translation, we used a 400 l reaction containing 10 g DNA, 50 mM Tris, pH 7.6, 5 mM MgCl2, 0.02 mM each dATP, dTTP and dGTP, 0.2 mM Cy3- or Cy5-dCTP (Amersham), 0.2 U DNA Polymerase I (Gibco BRL), and 30 l DNAseI/PolI enzyme mix (Gibco BRL). We incubated the reaction at 15 C for 60 min after which we stopped the reaction by incubation at 70 C for 10 min. We removed unincorporated nucleotides using a Sephadex G-50 spin column. We labeled genomic DNA by random priming in a 100 l reaction containing 0.003 - 0.6 g DNA, 1x random primers solution (BioPrime DNA Labeling System, Gibco BRL), 1 mM Tris, pH 7.6, 0.1 mM EDTA, 0.2 mM each of dATP, dTTP and dGTP, 0.1 mM dCTP, 0.4 mM Cy3 or Cy5-dCTP (Amersham) and 160 U Klenow fragment (BioPrime DNA Labeling System, Gibco BRL). We incubated the DNA with the random primers solution at 100 C for 10 min in a total volume of 84 l, prior to adding the other reagents and then incubated the 100 l reaction overnight at 37 C. We removed unincorporated nucleotides using a Sephadex G-50 column. <b>Hybridization.</b> We combined test and reference DNAs (~2 g of input genomic DNA for nick translation or ~0.6 g of input genomic DNA for random prime labeling) with Cot-1 DNA (80-100 g; Gibco BRL) and precipitated them with ethanol. 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After a 30 min incubation at room temperature, we opened the chamber, removed approximately three-quarters of the blocking solution, added the denatured and re-annealed hybridization mixture and then re-sealed the chamber. We placed the arrays on a slowly rocking table (~1 rpm) at 37 C to allow hybridization to occur over 16-72 h. After hybridization, we rinsed off the excess hybridization fluid with PN buffer (PN: 0.1 M sodium phosphate, 0.1% nonidet P40, pH 8), then washed once in 50% formamide, 2 x SSC, pH 7 at 45 C for 15 min, and finally in PN buffer at room temperature for 15 min. After draining excess liquid from the arrays, we mounted them in a solution containing 90% glycerol, 10% PBS and 1 M DAPI, and then sealed them with a cover slip. We drained excess glycerol-DAPI solution onto a paper tissue. <b>Imaging and analysis.</b> We acquired 16 bit 1024x1024 pixel DAPI, Cy3 and Cy5 images using a custom built CCD camera system, although we have also used commercial laser scanners for this purpose. We used ?UCSF SPOT? software (A.N.J. manuscript submitted) to automatically segment the spots based on the DAPI images, perform local background correction and to calculate various measurement parameters, including log2ratios of the total integrated Cy3 and Cy5 intensities for each spot. We used a second custom program SPROC to associate clone identities and a mapping information file with each spot so that the data could be plotted relative to the position of the BACs on the September, 2000 freeze of the draft human genome sequence (http://genome.ucsc.edu). SPROC also implements a filtering procedure to reject data based on a number of criteria, including low reference/DAPI signal intensity and low correlation of the Cy3 and Cy5 intensities with a spot. The SPROC output consists of averaged ratios of the triplicate spots for each clone, standard deviations of the triplicates and plotting position for each clone on the array, as well as other clone information stored in the database, such as STS content (Tables A and B). We edited the data files to remove ratios on clones for which only one of the triplicates remained after SPROC analysis and/or the standard deviation of the log2ratios of the triplicates was > 0.2. Keywords: other
2002-02-12 | GSE16 | GEO
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2019-07-02 | GSE133625 | GEO
Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.
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2013-07-04 | GSE30457 | GEO
Project description:Pretreated human follicular fluid was prepared for targeted metabolomics experiments. For this purpose, 12-point standard calibration curves in the respective range with reference material were measured, and the limit of detection (LOD) and limit of quantification (LOQ) for each neurotransmitter under investigation were calculated. LC-MS/MS was conducted by an ultra-performance liquid chromatography method using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm, Waters, USA). The Waters acquisition UPLC was coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, USA). The mobile phase A buffer was 10% methanol in water (containing 0.1% formic acid), and the mobile phase B buffer was 50% methanol in water (containing 0.1% formic acid). The LC gradient elution was at a flow rate of 0.3 mL/min at 0-8.0 min and 0.4 ml/min at 8.0-17.5 min, then 10%-30% B at 0-6.5 min; 30%-100% B at 6.5-7 min; 100% B at 7-14min; and 100%-10% B at 14-17.5min. The injection volume was 5 µl and the column temperature was set to 40 °C. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr:Tyrosine; Trp:Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid;HisA:Histamine; PA:Picolinic acid;TyrA:Tyramine;DA:Hydroxytyramine hydrochloride; TrpA:Tryptamine;5-HT:Serotonin hydrochloride;E:Adrenaline hydrochloride;KynA:Kynurenic acid;5-HIAA:5-Hydroxyindole-3-acetic acid;DOPA:Levodopa;XA:Xanthurenic acid;VWA:Vanillymandelic Acid; 5-HTTP:5-Hydroxytryptophan;MT:Melatonine)were simultaneously reported in LC-MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with Analyst 1.6 software.
2022-08-12 | PXD031560 | Pride