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Looking for changes in lipid levels with knock out of ACADM (shACADM) (part II)

ABSTRACT: GSC 8-11 cells (20 million) were transduced with lentivirus coding for shACADM 4, 6, or scr (control). ACADM is an enzyme responsible for the metabolism of medium chain fatty acids (C4-C12). A round of selection was performed using puromycin to ensure only cells that had been transduced survived. Knockdown of ACADM was confirmed via western blot. Pellets were collected by centrifuging cells for 10 min at max speed. The pellets were washed and each sample was split into quadruplicate.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Cultured Cells

SUBMITTER: Maureen Kachman

PROVIDER: ST000819 | MetabolomicsWorkbench | Fri Jul 14 00:00:00 BST 2017

REPOSITORIES: MetabolomicsWorkbench

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Looking for changes in lipid levels with knock out of ACADM (shACADM)

Project description:GSC 8-11 cells (20 million) were transduced with lentivirus coding for shACADM 4, 6, or scr (control). ACADM is an enzyme responsible for the metabolism of medium chain fatty acids (C4-C12). A round of selection was performed using puromycin to ensure only cells that had been transduced survived. Knockdown of ACADM was confirmed via western blot. Pellets were collected by centrifuging cells for 10 min at max speed. The pellets were washed and each sample was split into quadruplicate.

2017-06-19 | ST000771 | MetabolomicsWorkbench

Metabolomics Workbench ST000819 - GNPS - Looking for changes in lipid levels with knock out of ACADM (shACADM) (part II)

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Shotgun analysis of proteins in NDUFA11 KO and WT HEK 293T cells.

Project description:The effect of the depletion of an ETC complex I protein component - NDUFA11 on the proteome in HEK 293T cells was studied. TMT-based relative quantification of protein levels were performed in NDUFA11 KO vs WT cells and in NDUFA11 KO vs WT cells that expressed an EGFP-MAPT (Tau protein) construct. In this dataset relative levels of proteins in total cell extracts and in the soluble fraction (post centrifugation at 125,000 g, at which speed protein aggregates were isolated as pellets) were measured. This dataset is directly related to the dataset PXD031374 (protein aggregates isolated as pellets post centrifugation at 125,000 g).

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Metabolomics analysis of mouse liver with or without SIRT5 deficiency

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Real-time quantitative PCR analysis of Chromatin-associated snoRNA in human NB4 cells using Arraystar_384 platform

Project description:10 million cells were used for each sample. The cells were pre-treated with or without Doxorubicin (2µM for 4h) before collection and washed with ice-cold 1 × PBS. The nuclei pellet was further fractionated into nucleoplasmic fraction (supernatant) and chromatin pellet by using MWS buffer. All the buffers above were supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and Ribonuclease Inhibitors (Promega). The RNA precipitation solution (RPS) buffer was added into the cytoplasmic fraction and nucleoplasmic fraction immediately and each fraction was stored at -20°C for at least 1h. The two fractions were centrifuged and the resulting pellets were washed with 75% ethanol. All the pellets from the chromatin fraction, nucleoplasmic fraction and cytoplasmic fraction were added with 1mL TRIzol (Invitrogen) to extract RNA. The RNA pellet in TRIzol was fully dissolved by adding EDTA to the final concentration of 5mM and heat it to 65°C. The resulting RNAs from each fraction were subjected to DNase I (Sigma-Aldrich) treatment followed by phenol/chloroform extraction to remove any contaminant DNA. The RNA pellets of each fraction were then dissolved in equal volumes of RNase-free water and used for snoRNA qRT-PCR analyses.

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Whole Exome Sequencing Data of Dabrafenib Resistant HT-29 Cell Line

Project description:HT-29 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib resistant derivatives of these cell lines were established, respectively. Five million barcoded HT-29 cells were seeded into 15 cm cell culture dishes. When the cells reached confluency, two million cells per dish were seeded into four different 15 cm dishes (DMSO Control, Replica A, B, C) and two million cell pellets were stocked as initial cell population. Harvesting used medium through the experiment was performed at monthly intervals. Barcoded HT-29 cell line replicates A, B, and C were treated with 2XIC50 (199.6 nM) of dabrafenib concentration for the duration of 3 months.Barcoded data can be accessed via accession code E-MTAB-13018. Whole exome sequencing of dabrafenib-resistant A replicate and DMSO control cell lines were carried out.

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Tracking of barcodes introduced in the genomes of HCC827 cell populations over time under high doses of gefitinib and trametinib

Project description:HCC827 cells were barcoded using the ClonTracer lentiviral barcode library such that the majority of cells were infected with a single barcode. One million cells were expanded to ~120 million cells and split into 8 HYPERfasks. Two HYPERfasks were grown under DMSO and grown until confluence. In six HYPERfasks cells were grown under a GI90 concentration of one of two different inhibitors, gefitinib and trametinib (3 HYPERfasks each). Cells achieved confluence at 4 and 9 weeks for gefitinib and trametinib respectively. During this time, the medium and inhibitor were replenished weekly and DNA was extracted from the medium to track barcode content from dying cells.

2020-08-18 | E-MTAB-8841 | biostudies-arrayexpress

Barcode Sequencing Data of Dabrafenib Resistant HT-29 Cell Line

Project description:HT-29 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib resistant derivative of these cells were established. Five million barcoded HT-29 cells were seeded into 15 cm cell culture dishes. When the cells reached confluency, two million cells per dish were seeded into four different 15 cm dishes (DMSO Control, Replica A, B, C) and two million cell pellets were stocked as initial cell population. Harvesting used medium through the experiment was performed at monthly intervals. Barcoded HT-29 cell line replicates A, B, and C were treated with 2XIC50 (199.6 nM) of dabrafenib concentration for the duration of 3 months. Following the end points of 3 months for each cell line, DNA isolation from harvested cell lines and collected medium of resistant A cell lines were carried out and barcodes were sequenced.

2023-09-06 | E-MTAB-13018 | biostudies-arrayexpress

Reanalysis of Pandey lab's draft of the human proteome

Project description:The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides was lacking. To this end, Akhilesh Pandey's lab reported a draft map of the human proteome based on high resolution Fourier transform mass spectrometry-based proteomics technology, which included an in-depth proteomic profiling of 30 histologically normal human samples including 17 adult tissues, 7 fetal tissues and 6 purified primary hematopoietic cells ( http://dx.doi.org/10.1038/nature13302 ). The profiling resulted in identification of proteins encoded by greater than 17,000 genes accounting for ~84% of the total annotated protein-coding genes in humans. This large human proteome catalog (available as an interactive web-based resource at http://www.humanproteomemap.org) complements available human genome and transcriptome data to accelerate biomedical research in health and disease. Pandey's lab and collaborators request that those considering use of this primary dataset for commercial purposes contact pandey@jhmi.edu. The full details of this study can be found in the PRIDE database: www.ebi.ac.uk/pride/archive/projects/PXD000561/. This ArrayExpress entry represents a top level summary of the metadata only which formed the basis of the reanalysis performed by Joyti Choudhary's team ( jc4@sanger.ac.uk ), results of which are presented in the Expression Atlas at EMBL-EBI : http://www.ebi.ac.uk/gxa/experiments/E-PROT-1.

2015-03-10 | E-PROT-1 | biostudies-arrayexpress

MAX-mutant small cell lung cancers exhibit impaired activities of MGA-dependent ncPRC1.6.

Project description:The MYC axis is commonly disrupted in cancer, mostly by activation of the MYC family of oncogenes, but also by genetic inactivation of MAX, the obligate partner of MYC, and of the MAX partner, MGA, both of which are members of the polycomb repressive complex, ncPRC1.6. While the oncogenic properties of the MYC family have been extensively studied, the tumor suppressor functions of MAX and MGA and the role of the MYC genes in MAX-mutant cells remain unclear. To address these knowledge gaps, we used chromatin immunoprecipitation, RNA-sequencing and mass spectrometry-based proteomic analysis in MAX-restituted and MYC oncogenic-transformed cell lines derived from human small cell lung cancer (SCLC), which is a high-grade neuroendocrine type of lung cancer. We found that MAX-mutant SCLC cells express ASCL1 and ASCL1-dependent targets, implying that these cells belong to the ASCL1-dependent group of SCLCs. In the absence of MAX, even after ectopic overexpression of MYC, we found no recruitment of MYC to the DNA. Furthermore, MAX reconstitution triggered pro-differentiation expression profiles that shifted when MAX and oncogenic MYC were co-expressed. Although ncPRC1.6 could be formed, the lack of MAX restricted global MGA occupancy, selectively driving its recruitment towards E2F6 motifs. Conversely, MAX restitution enhanced MGA occupancy and global gene repression of genes involved in different functions, including stem-cell and DNA repair/replication. Our data reveal that MAX-mutant SCLCs have ASCL1 characteristics, and are MYC-independent, and that their oncogenic features include deficient ncPRC1.6-mediated gene repression.

2021-09-22 | PXD017658 | Pride

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