MRNA Profiling of cytoplasmic and 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-56150 | 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 µM 4EGI-1 or DMSO. After 2 h incubation at 37°C 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 - 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 °C 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 °C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturer´s 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 µl of RNase-free water. RNA concentrations were determined and the samples were stored at -80 °C.
2014-01-01 | E-GEOD-41382 | biostudies-arrayexpress
Project description:Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2. Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using LipofectamineM-bM-^DM-" RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturerM-bM-^@M-^Ys recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x CompleteM-BM-. protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a BioruptorM-bM-^DM-" UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 M-bM-^AM-0C with rotation. Antibodies were immunoprecipitated with 60 M-BM-5l protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 M-bM-^AM-0C for 2 hours. Next, proteins and RNA were digested by adding 33 M-BM-5l of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 M-bM-^AM-0C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using QubitM-bM-^DM-" (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.
2013-09-28 | E-GEOD-50177 | biostudies-arrayexpress
Project description:While the regulation of metabolic enzymes by oncogenic drivers or tumor suppressors has been intensively studied over recent years, our understanding of how metabolic processes directly regulate cell proliferation has remained fragmentary. Here we show how the alteration of metabolism directly affects cell cycle progression in cancer cells. We found that activation of the nuclear receptor peroxisome-proliferation activated receptor gamma (PPARM-NM-3), a transcriptional master regulator of lipid metabolism, inhibits the growth of lung adenocarcinoma cells by triggering a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARM-NM-3-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. Both of these changes can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or M-NM-2-oxidation of fatty acids. Thus, we provide a mechanism that directly links metabolic changes to inhibition of cancer cell cycle progression by altering ROS levels. We generated PPARG-LAP BAC transgenic NCI-H2347 and NCI-H1993 cell lines using the BAC-transgenesis approach. Cells at 80% confluency (~1-1.5x107) were cross-linked with 1% formaldehyde for 10 minutes at 37M-BM-0C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4M-BM-0C, the cell pellets were resuspended in 100 M-NM-<l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4M-BM-0C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 12 minutes). The sheared chromatin with a fragment length of ~200 M-bM-^@M-^S 600 bp) was centrifuged at 10,000 g for 10 minutes at 4M-BM-0C). 100 M-NM-<l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4M-BM-0C overnight with 6 M-NM-<g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitations were carried out by incubating with 40 M-NM-<l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4M-BM-0C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 M-NM-<l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65M-BM-0C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (Qiagen) and eluted into 30 M-NM-<l H2O according to the manufacturerM-bM-^@M-^Ys protocol after treatment with RNase A and Proteinase K.
2014-09-25 | E-GEOD-58382 | biostudies-arrayexpress
Project description:We observed the expression profile of the total mRNA of wild-type and TTHA1559 disrupted Thermus thermophilus HB8 strain at 70M-BM-0C. Wild-type and TTHA1559 disrupted T. thermophilus HB8 strains were pre-cultured at 70M-BM-0C in 10 ml of synthetic medium. The cells (0.1 ml) were inoculated into 10 ml of the same medium and then cultivated at 70M-BM-0C. Cells were collected after 5h, and then crude RNA was extracted. The expression of each mRNA was analyzed on a GeneChip.
2014-08-03 | E-GEOD-33985 | biostudies-arrayexpress
Project description:The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOHM-bM-^@M-^Ys modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). SUDHL-2 and SUDHL-4 diffuse large B-cell lymphoma (DLBCL) cell lines (ATCC) were cultured in RPMI Medium 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) at 37M-BM-0C in 5% CO2. EtOH-treated cells were cultured in sealed flasks to maintain EtOH concentrations, 20mM in the culture medium, water was added to EtOH control wells. INK128-treated cells were treated with INK128 at 40nM (Selleckchem) dissolved in dimethyl sulfoxide (DMSO) or 0.01% DMSO was added for the control wells. To create polysomal fractions, cells were lysed in cytoplasmic lysis buffer (5mM Tris, 2.5mM MgCl2, 1.5mM KCl, 1% Triton X-100, 0.5% Sodium Deoxycholate and 2 mM DTT), loaded on 10 - 50% linear sucrose gradients and fractionated. The RNA in each fraction was monitored by optical density measurement (A254) and eleven fractions were collected with a fraction collector (Brandel). The RNA from each fraction was isolated by Trizol (Invitrogen) and used for RT-qPCR analysis. RNA from high molecular weight polysomal fractions, with actively translated mRNAs (fractions 9 - 11) were pooled and used for microarray analysis. RNA was isolated using Qiagen RNeasy protocols and quality and quantity were checked with an Agilent 2100 Bio-Analyzer using RNA 6000 Nano chips. Labeling and amplification were done with the standard Illumina protocol and the biotin-labeled cRNA was hybridized to Illumina's HumanHT-12 V4.0 Expression BeadChip. The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3.
2015-03-03 | E-GEOD-62790 | biostudies-arrayexpress
Project description:We observed the expression profile of the total mRNA of litR (TTHB100) deletion mutant of T. thermophilus HB8 strain grown in a rich (TM) medium at 60 M-BM-0C under the dark condition compared with that of wild-type. Three wild-type strain of T. thermophilus HB8 were pre-cultured at 60 M-BM-0C for 16 hours in 100 mL of TM medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.5 with NaOH. The cells (1 mL) were inoculated into 100 mL of the synthetic medium and then cultivated at 60M-BM-0C for 8 hours under dark condition. Then the 1.0 mM of ZnSO4 was added into the medium, and continued cultivation. Cells were collected, and then, the expression of each mRNA was analyzed.
2014-08-17 | E-GEOD-31463 | biostudies-arrayexpress
Project description:In order to find function of PfmR protein from T. thermophilus HB8, we observed the expression profile of the total mRNA of the PfmR-deficient strain grown in a rich medium at 70M-BM-0C for 360 min. Three PfmR-deficient T. thermophilus HB8 strains were each pre-cultured at 70M-BM-0C for 16 h in 4 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (1.6 ml) were inoculated into 1 liter of the same medium and then cultivated at 70M-BM-0C. Cells were collected after 360 min, and then, the expression of each mRNA was analyzed.
2012-10-02 | E-GEOD-36881 | biostudies-arrayexpress
Project description:We analyzed the expression profile of wild-type strain and ttha1564 deletion mutant strain of Thermus thermophils HB8 under alkylating condition. In wild type strain, 107 genes and 96 genes were over 2-fold up- and under 0.5-fold down-regulated by alkylation, respectively. In ttha1564 deletion mutant, 35 genes and 12 genes were over 2-fold up- and under 0.5-fold down-regulated by alkylation, respectively. Keywords: stress response comparison The T. thermophilus HB8 wild-type and delta ttha1564 strains were cultured in TT medium (0.4%(w/v) polypeptone, 0.2%(w/v) yeast extract, 0.1%(w/v) NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, (pH 7.5)) at 70M-BM-0C until the OD600 reached 0.8. The cultures were then divided into two flasks and an equal volume of TT medium prewarmed at 70M-BM-0C was added. One of the media culture of each strain contained 100 micro g/ml MNNG. We confirmed that the cells can continue to grow in TT medium containing 100 micro g/ml MNNG but DNAs are alkylated sufficiently (Onodera, 2011). After further cultivation at 70M-BM-0C for 10 min, the diluted cultures were collected into an equal of 100% ethanol and stored at -80M-BM-0C. The procedures used for extraction of crude RNA, synthesis of cDNA, labeling of cDNA fragment, hybridization to a TTHB8401a520105F GeneChip (Affymetrix) and data analysis were basically the same as those described previously (Agari et al., 2008; Shinkai et al., 2007). To determine the mRNA expression level of each sample, image data for the three independent samples were processed. stress response comparison
2012-03-08 | E-GEOD-27818 | biostudies-arrayexpress
Project description:We observed the expression profile of the total mRNA of the wild-type strain grown in a rich medium at 70 deg-C for 360 min. Three wild-type T. thermophilus HB8 strains were each pre-cultured at 70M-BM-0C for 16 h in 4 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70M-BM-0C. Cells were collected after 360 min, and then, the expression of each mRNA was analyzed.
2012-10-02 | E-GEOD-36883 | biostudies-arrayexpress
Project description:We observed the expression profile of the total mRNA of PfmR (TTHB023) deletion mutant of Thermus thermophilus HB8 strain grown in a rich (TT) medium at 70M-BM-0C compared with that of wild type. Seven wild-type and three mutant T. thermophilus HB8 strains were each pre-cultured at 70M-BM-0C for 16 h in 4 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells were inoculated into 1 liter of the same medium and then cultivated at 70M-BM-0C. Cells were collected after 360 min, and then crude RNA was extracted. In order to compare mRNA expression in the PfmR deletion mutant with that of wild type, the expression of each mRNA was analyzed on a GeneChip.
2013-03-18 | E-GEOD-36909 | biostudies-arrayexpress