Project description:To identify differentially up or downregulated genes in MCF7_ADR cell compare to MCF7, we have employed whole genome microarray expression profiling. We used microarray to evaluate the up or down regulated genes in doxorubicin resistant MCF7_ADR cells compare to MCF7
Project description:The purpose of the study was to identify mRNA bound to HuR in the presence of doxorubicin in MCF7 cells. We collected cytoplasmic RNA from untreated and treated cells and detected differentially expressed genes (DEGs). We also coimmunoprecipitated HuR and IgG (as control) from doxorubicin treated cells. Comparison between HuR RIP and IgG RIP signals was used to discriminate specific mRNA bound to HuR. HuR coimmmunoprecipitated material was hybridized together with cytoplasmic mRNA of doxorubicin treated cells, enabling the fold enrichment calculation and the selection of mRNAs bound to HuR. Keywords: RIP-Chip, HuR, doxorubicin, MCF7, HuR consensus binding, post-transcriptional regulation. We subjected MCF7 cells to starvation for 24h and then we added doxorubicin at final concentration of 10 uM, profiling before and after 4 hours of treatment in biological quadruplicate (only on cytoplasmic mRNAs, as HuR was found in the cytoplasm). Differentially expressed genes, altered during the treatment, were identified. Data derived from HuR RIP-Chip and IgG RIP-Chip (in biological quadruplicate) allowed the identification of specific mRNAs bound to HuR. The comparison between HuR RIP-Chip and cytoplasmic extracts from doxorubicin treated cells (in biological triplicate) identified those genes that were more strictly bound to HuR independently from their expression levels.
Project description:The effects of several compounds on the MCF7 human adenocarcinoma mammary cell line were analysed by gene expression profiling. Tested compounds: HSP90 inhibitors: 17AAG (Tanespimycin), NVP-AUY922, NMS-E973 (cpd developed at NMS). CDK inhibitors: CDK-887 (cpd developed at NMS). Topoisomerase inhibitors: Doxorubicin, SN38 (active metabolite of Irinotecan). The MCF7 cell line was treated with the different compounds for 6 hours at a dose equal to 5 times the IC50. Untreated MCF7 cells were used as a control. Two replicates per treatment.
Project description:Transcriptional elongation factor Elongin A exhibits an activity to repress the temporary pause of RNA polymerase II (RNAPII) on the DNA template, resulting in increase of mRNA synthesis in vitro. However, actual elongation ability of Elongin A in mammalian cells has not been determined yet. To elucidate the practical transcriptional role of mammalian Elongin A in vivo, we carried out whole genome expression analysis using Elongin A knocked-down HeLa cells. Gene expression in Elongin A knocked-down HeLa cells was measured at 2 hours after doxorubicin treatment (1microM).
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.