Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA168022 | ENA |
REPOSITORIES: ENA
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Project description:1. Quantitative Proteomics: MDA-MB-231, MDA-MB-468, and MCF12A cells were treated with DMSO (vehicle control) or SU056 (novel small molecule drug candidate). Quantitative proteomics analysis was performed on cell lysates. 2. Cellular Thermal Shift Assay (CETSA): MDA-MB-231 cells were treated with DMSO or SU056 and incubated at different temperatures and protein differences in the resulting soluble and insoluble fractions were determined.3. Cellular Thermal Shift Assay (CETSA): MDA-MB-231 YBOX1 KD cells were treated with DMSO or SU056 and incubated at different temperatures and protein differences in the soluble fractions were determined.
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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived CTU-treated MDA-MB-231 cell's transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Purified RNA of CTU-treated MDA-MB-231 cells (10 μM, 6 h) were sent to the Australian Genome Research Facility for RNA-Seq analysis. Differential expression analysis was performed by edgeR and pathway enrichment was performed using the Reactome database as a reference.qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: RNA-Seq profiling identified a total of 14,951 genes in MDA-MB-231 cells, and detected 382 differentially expressed genes greater than or equal to (≥) 2-fold after 10 μM CTU treatment at 6 h. Genes whose expression was changed significantly were identified using the false discovery rate (FDR), an adjusted P-value for multiple testing. The expression of 250 and 132 genes was significantly up-regulated and down-regulated, respectively, to ≥2-fold of control in CTU-treated cells (FDR<0.05). Secondary pathway analysis was performed using the Reactome database to identify the principal biological pathways that were altered in CTU-treated MDA-MB-231 cells. This analysis implicated several mechanisms, including disruption of the tumour microenvironment, activation of cellular stress signalling and activation of cell cycle arrest. ER stress via the UPR survival mechanism was a major dysregulated pathway in MDA-MB-231 cells that had been treated with CTU (10 μM, 6 h) compared with control (FDR= 0.0001). Conclusions: Our study represents the first detailed analysis of CTU-treated MDA-MB-231 cells (10 μM, 6 h) l transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
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