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Effects of environmental chemicals on development of neuronal cell lineages derived from mouse ES cells

ABSTRACT: This study provides a comprehensive evaluation of changes in gene expression during treatment with environmental chemicals in embryonic bodies derived from mouse embryonic stem (ES) cells. ES cells were maintained under the feeder cells in phenol red-free DMEM Medium (supplemented with 15% FBS, 100uM Non-essential amino acids (NEAA), 1000U/ml Leukemia inhibitory factor(LIF), 100uM 2-mercaptoethanol(2-ME), and 0.5% penicillin/streptomycin). Cells were gently washed in warm PBS and transferred to phenol red-free DMEM Medium (supplemented with 15% KnockOut Serum Replacement, 100uM NEAA, 100uM 2-ME, and 0.5% penicillin/streptomycin) in Microsphere array (MSA300F) and exposed with low and high levels of each chemical just after removing LIF from the culture media. Prior to collection, cells were washed in warm PBS, resuspended and briefly incubated in the QIAGEN RLT buffer, and finally collected in triplicate at 48 hours. Following RNA isolation, the best RNA yields for each replicate set was selected for target preparation and microarray processing.

ORGANISM(S): Mus musculus

PROVIDER: GSE18503 | GEO | 2009/10/10

SECONDARY ACCESSION(S): PRJNA120253

REPOSITORIES: GEO

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Project description:In this project, we employed a dynamic mass spectrometry-based proteomic approach to obtain global maps of basal autophagic flux in human primary fibroblasts (HCA2-hTert). The data provide a comparison of protein degradation rates between dividing and quiescent HCA2-hTert cells. To further investigate the role of autophagy in up-regulated protein degradation, protein degradation rates were also measured in autophagy-deficient (Atg5-/-) in both dividing and quiescent cells. Steady-state protein level changes in dividing and quiescent cells were measured by standard SILAC. To investigate the role of PSME1 in proteasome activity regulation, protein degradation rates in PSME1 knockdown cell line were measured by SILAC. Stable isotope labeling 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. Both wildtype and Atg5-/- Cells were gradually adapted to this media and prepared for labeling experiments. Cells were then plated at a density of 500,000 cells per 10 cm plate. One day after plating, the dividing 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). Cells were collected after 0, 1, 2, and 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 8 days after plating, the confluent quiescent 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). Cells were collected after 0, 2, 4, and 6 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. In order to measure changes in steady-state protein levels by standard SILAC, WT cells were gradually adapted to Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Then, WT cells were passaged in MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13 C6, 99%) and L-lysine:2HCl (13 C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo scientific) for eight passages. Cells were then plated at a density of 500,000 cells per 10-cm plate. 2 days after plating, dividing cells were collected and washed with PBS, and cell pellets were frozen prior to further analysis. Following extraction (see below), equal protein amounts of dividing and quiescent WT cells were mixed before mass spectrometric analysis. To measure protein degradation in PSME1 knockdown cell line, 8 days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13D4, 96%-98%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific). Cells were collected after 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis.

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Effects of environmental chemicals on development of neuronal cell lineages derived from mouse ES cells

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