Dataset Information

Proteomics of Matrigel, LC-MS/MS

ABSTRACT: ECM is the physicochemical support for the cells living in the tissue microenvironment. it is important to know the protein contents within the ECM that are critical for extra- and intracellular signaling, cell growth, migration, cell-cell/ECM interactions. The ECM gel derived from the Engelbreth-Holm-Swarm murine sarcoma (commonly known as Matrigel or lrECM) purchased from the Sigma Aldrich in the liquid concentration of 8-12 mg/ml (Lot # 064M4075V) was used for the LC-MS/MS analysis and to prepare the 3D scaffolds used for downstream experiments. The Matrigel mass spectrometry data will be used to compare with those of the decellularized mice mammary tissue ECM/DBT-TMS.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skin

SUBMITTER: Weimin Li

LAB HEAD: Weimin Li

PROVIDER: PXD005696 | Pride | 2018-10-26

REPOSITORIES: Pride

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Publications

A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening.

Rijal Girdhari G Li Weimin W

Science advances 20170913 9

Most of the anticancer drug candidates entering preclinical trials fail to be approved for clinical applications. The following are among the main causes of these failures: studying molecular mechanisms of cancer development, identifying therapeutic targets, and testing drug candidates using inappropriate tissue culture models, which do not recapitulate the native microenvironment where the cancer cells originate. It has become clear that three-dimensional (3D) cell cultures are more biologicall ...[more]

PMID: 28924608

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Project description:This study was undertaken to identify how gene expression of the RT4 bladder papilloma cells respond to extracellular matrics (ECM) Matrigel. Gene expression profile of RT4 cells grown on Plastic was compared with expression profiles of RT4 cells grown on Matriges over 12 hours-9 days timecourse. Total 5881 hypervariable (HV) genes were found to vary significantly over the time points, indicating a response to matrix change. Correlational clustering showed these HV genes fell into distinct families, predominantly genes which became highly expressed on ECM. Another cluster show genes expressed at 12 hours after grown on Matrigel and third cluster show genes whose expression decreased below noise level on Matrigel. This indicate that processes responsible for cell adaptation to ECM happen within first 12 hours and the second cluster form last response of those active processes. Over 1-9 days of cell grows on Matrigel those genes are constantly expressed. Cell signaling and cellular growth and proliferation accounted for major functions for those genes. G-protein signaling, immune response and NF-kB signaling accounted for most dramatically affected canonical pathways. Genes sharing similar ontologies also share several overrepresented transcription regulatory elements confirmed by independent experiment on Panomics platform.Thus, rapid response for ECM orchestrated by several transcription factors was observed. Identified canonical pathways indicate possible targets for manipulating cell phenotype on ECM. Keywords: extracellular matrix, gene expression, RT4, Matrigel, microarray, time course Three dimensional gel cultures with Matrigel were performed by layering 0.8 mls of ice cold Matrigel onto polyethylene terephthalate membranes of 6-well cell culture inserts (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ). Gels were solidified at 37Âº C. RT4 cells were trypsinized, brought up on 20 mls McCoys media with 10% Fetal Calf Serum (FCS). Cells were centrifuged then brought up in McCoys media with 10% FCS to a concentration of 500,000 cells/250Âµl. 250Âµl added on top of each solidified matrigel disc and 2mls of McCoys with 10% FCS layered underneath each culture insert. Cells were maintained for 10 days and media replenished underneath the insert twice a week. Cells were harvested at 12 hrs, 24hrs, 2 days, 3 days, 4days, 6 days, 7 days, 8 days, and 9 days of growth in culture using Matrisperse (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) and RNA collected. Data were normalized as was described previously in Dozmorov I et al., Nucleic Acids Res. 2004 Oct 28;32(19). In general, intensities were converted to expression and normalized to low-expressed genes, which provide possible technical noise level. The arrays are then adjusted to each other by robust linear regression and this Log10 transformed data presented for each sample. Group of low-expressed genes is selected by normal distribution fitting. This group provides internal standard of measurement noise (ISMN). Genes above 5SD from mean of ISMN were selected as expressed

Dataset Information

Proteomics of Matrigel, LC-MS/MS

Publications

A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening.

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