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Comparison of gene expression profiles of miR-142-/- primary megakaryocytes and WT primary megakaryocytes

ABSTRACT: Whole fetal livers were collected from mouse fetuses at embryonic day 14.5 (E14.5), and single-cell suspensions were prepared by successive passage through 18-, 21 and 23-gauge needles. Fetal liver cells were maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO; Peprotech). After 5 days of culture, megakaryocytes were purified using a discontinuous bovine serum albumin gradient (BSA, Sigma–Aldrich; 3%, 1.5%, and 0%). Total RNA was isolated with Tri–Reagent (MRC) following manufacturer’s instructions, and its quality was assessed with ND–1000 Nanodrop (Peqlab) and on a 1.5% agarose gel.

ORGANISM(S): Mus musculus

PROVIDER: GSE52141 | GEO | 2014/06/11

SECONDARY ACCESSION(S): PRJNA227016

REPOSITORIES: GEO

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Comparison of gene expression profiles of miR-142-/- primary megakaryocytes and WT primary megakaryocytes

Project description:Whole fetal livers were collected from mouse fetuses at embryonic day 14.5 (E14.5), and single-cell suspensions were prepared by successive passage through 18-, 21 and 23-gauge needles. Fetal liver cells were maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100M-BM-5g/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO; Peprotech). After 5 days of culture, megakaryocytes were purified using a discontinuous bovine serum albumin gradient (BSA, SigmaM-bM-^@M-^SAldrich; 3%, 1.5%, and 0%). Total RNA was isolated with TriM-bM-^@M-^SReagent (MRC) following manufacturerM-bM-^@M-^Ys instructions, and its quality was assessed with NDM-bM-^@M-^S1000 Nanodrop (Peqlab) and on a 1.5% agarose gel. Gene expression was measured in primary megakaryocytes cultured from miR-142-/- fetal livers and wild type fetal livers at E14.5. Two independent experiments were performed.

2014-06-11 | E-GEOD-52141 | biostudies-arrayexpress

Comparison of gene expression profiles of p45-/- primary megakaryocytes and WT primary megakaryocytes

Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells.

2013-08-07 | GSE35645 | GEO

Comparison of gene expression profiles of p45-/- primary megakaryocytes and WT primary megakaryocytes

2013-08-07 | E-GEOD-35645 | biostudies-arrayexpress

Effect of hydrogen peroxide on Echinococcus granulosus protoescoleces proteome

Project description:We collected protoescoleces (PSCs) from individual bovine cysts. Then we incubated 20 uL of PSCs in RPMI medium supplemented with 10% fetal bovine serum with 2.5 mM hydrogen peroxide (treated) or without hydrogen peroxide (Control) for 2 h at 37 °C. After that we collected PSCs by sedimentatios in 1.5 mL tube and washed 4 times with PBS at 37°C.

2020-05-27 | PXD015801 | Pride

A gene expression signature of PDGF-stimulated fibroblasts

Project description:Analysis of platelet-derived growth factor (PDGF)-stimulated fibroblasts. Results provide insight into novel pro-tumorigenic factors induced by PDGF-activation of fibroblasts. 1.2 x 10e6 BJ-hTERT fibroblasts were cultured in 10cm dish with Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies, Gergy-Pontoise, France) containing 1% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 ng/ml) at 37ÂºC in a 5% CO2 humidified atmosphere during 24h. Cultures were stimulated with or without 20ng/mL PDGF-BB (Peprotech, New Jersey, USA) for another 24h prior to harvest. Unstimulated fibroblasts were used as control samples. Cultures of both unstimulated and stimulated fibroblasts were done in triplicate.

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Time-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing

Project description:In this project, a more efficient methodology for obtaining multi-timepoint kinetic data for proteome-wide analyses of protein turnover is developed and used to globally measure the kinetics of protein turnover in primary human fibroblasts (HCA2-hTert). This approach takes advantage of the multiplexing capabilities of isobaric tandem mass tags (TMT) to measure fractional SILAC labeling of multiple time-points in a single LC-MS/MS run. Human dermal fibroblasts (HCA2-hTert) (19, 20) were maintained in Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 U/mL streptomycin at 37℃ with 5% CO2. The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were gradually adapted to the labeling media and were then plated at a density of 500,000 cells per 10 cm plate. For dividing cells, one day after plating, the cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific) and were subsequently collected after 0, 24, 48, 72 hours of labeling. For quiescent cells, the cultures were grown for 8 days to achieve a state of quiescence by contact inhibition. The confluent quiescent cultures were switched to the labeling media and cells were collected after 0, 6, 12, 24, 36, 48, 72, 96, 144, 192, 336 hours of labeling. All cells were washed with PBS and frozen as cell pellets prior to further analysis.

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AKT/PACS2 participates in renal vascular hyperpermeability by regulating endothelial fatty acid oxidation in diabetic mice

Project description:HUVECs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in an Endothelial Cell Medium (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were treated with 30 mM glucose and 0.1 mM palmitic acid (P0500, Sigma-Aldrich, USA) dissolved in 0.5% bovine serum albumin (BSA) for 48 h to simulate HGHF treatment. The Akt inhibitor MK-2206 2HCl was purchased from Selleck Chemicals (S1078, Houston, TX, USA).

2022-08-12 | PXD033227 | Pride

Mouse primary megakaryocytes, wild type & NF-E2p45-deficient

Project description:To identify p45 target genes, we conducted gene expression profiling with p45-null megakaryocytes cultured from E14.5 fetal liver. Many genes encoding membrane proteins and enzymes related to platelet function, including Txas, Glycoprotein 6 (Gp6) and Selectin P (Selp), were repressed in the absence of p45. Considering the similar DNA binding specificity of p45 and Nrf2 in vitro, we expected p45 to activate cytoprotective genes that are established Nrf2 targets. However, the expression of numerous detoxifying enzymes and stress-responsive genes, including NAD(P)H:quinone oxidoreductase (Nqo1), were increased in the absence of p45. To obtain wild type and p45-null fetal livers, p45+/- mice were crossed. Whole livers were recovered from mouse fetuses at E14.5 and single cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako) supplemented with 20% charcoal-stripped fetal bovine serum, 50 units/ml penicillin, 50 Âµg/ml streptomycin, and 50 ng/ml of recombinant human thrombopoietin. CD41+ cells were selected from a 4 day primary culture of E14.5 fetal liver cells using a MACS magnetic system (Miltenyi Biotec). The fetal liver culture with TPO described above was incubated with FITC-conjugated anti-CD41 antibody (BD Pharmingen, clone MWReg30) followed by incubation with anti-FITC microbeads. Subsequently, the microbeads were selected magnetically through MACS large cell columns (Miltenyi Biotec). Total RNA was extracted from the CD41+ cells using Isogen (Nippon Gene).

2010-03-22 | E-GEOD-15581 | biostudies-arrayexpress

mRNA transcriptome sequencing of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) from Lyz2-Cre;Trpv2fl/fl and Trpv2fl/fl mice.

Project description:Femurs and tibias were dissected from Trpv2fl/fl and Lyz2-Cre;Trpv2fl/fl mice. Bone marrow was flushed from the bones, and the red blood cells were lysed with ACK lysis buffer. Debris was removed by passing cells through a 70 μM strainer and cells were counted via Countess II FL Automated Cell Counter. Approximately 5 × 106 bone marrow cells were cultured in 100 mm dishes in DMEM (Thermo Fisher Scientific, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% streptomycin-penicillin at 37 °C in a humidified incubator gassed with 5% CO2. GM-CSF (20 ng/mL, Peprotech) and M-CSF (10 ng/mL, Peprotech) were added to the bone marrow cultures for differentiation of BMDCs and BMDMs, respectively. The medium was changed every 3 days. On day 7, cells were collected and homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis. TruSeq Stranded mRNA LT Sample Prep Kit was used to synthesized the first strand cDNA through the action of random primers and reverse transcriptase, the second strand cDNA is synthesized with the first strand cDNA as the template, cDNA library construction followed by sequencing on an Illumina NovaSeq 6000 platform. RNA-sequencing reads were first trimmed to remove poly(A) and unqualified reads with cutadapt, then aligned to Mus musculus GRCm38 genome with Ensembl. The numbers of counts were summarized at the gene level using HTseq. Gene expression values were computed from fragments per kilo bases per million fragments (FPKM) values produced by addition of a pseudocount of 1 and log2 transformation of the results.

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