Full transcriptome analysis of MCF-7 cells treated with screening hit compounds versus control
ABSTRACT: This experiment was performed as part of a hit screening project. Transcriptome analysis was performed to determine whether the screened hits induced a gene expression pattern consistent with proteasome inhibition. MCF-7 cells were exposed to 2x[IC50] of hit compounds for six hours. RNA was processed according to manufacturer's protocol for preparation of HuGene 2.0ST chips to acquire biotinylated sense-strand DNA. Biotinylated DNA was hybridized to HuGene 2.0ST chips at 37°C for 16 hours in a GeneChip® Hybridization Oven 645, washed and stained using the GeneChip® Fluidics Station 450 using the FS450_0002 protocol and scanned with a GeneChip® Scanner 3000 7G.
Project description:This experiment is a part of the larger study on angiotensin II role in the upregulation of the CX3CL1 expression and its release in human first trimester placenta. CX3CL1 (or fractalkine) is chemokine involved in the pathogenesis of hypertension, which can be secreted by trophoblasts, initiating the cross talk with monocytes. Thus, we performed the microarray experiment to study the effects of recombinant human CX3CL1 on the transcriptome of human primary monocytes.
Project description:Celecoxib is recognized to have chemopreventive and anti-cancer property beyond its anti-inflammatory function. Celelcoxib treatment in AGS cells down regulates Wnt/β-catenin, STAT3, RXR and ERK/MAPK signaling pathways. Transcriptional profiling after celecoxib shows differential regulation of oncogenic pathway regulated genes. AGS cell were treated with celecoxib in triplicates and incubated for 24 hours. The processed samples were hybridized in “HuGene-1_0-st” array and the differentially expressed genes were taken for pathway analysis and transcription factor enrichment analysis.
Project description:Global gene expression in TET2 mutant and Wild type patients. We performed an integrated analysis of global DNA methylation and gene expression data to investigate the effects of DNA hypermethylation on gene expression. Total RNA was isolated from 4 TET2mut and 5 TET2wt were isolated using standard RNAeasy kit protocol (Qiagen). Their gene expression profiles were obtained using the[HuGene-1_0-st] Affymetrix.
Project description:The chromatin of individual chromosomes is organized into chromosome territories (CTs) in the interphase nucleus. The spatial arrangement of CTs is non-random and evolutionarily conserved. The gene-dense and gene-poor CTs are positioned in the nuclear center and periphery, respectively. As candidates for key molecules involved in nuclear organization, we have investigated the nuclear actin-related proteins (Arps), which include the evolutionarily conserved actin-family together with conventional actin. We used a conditional knockout system with chicken DT40 cells to analyze the functions of the actin-related protein Arp6. Consistent with a previous identification of Arp6 in the SRCAP chromatin remodeling complex, the histone variant H2AZ was significantly decreased in the chromatin of Arp6-deficient cells. Most importantly, Arp6-deficient cells had impaired radial positioning of both gene-poor macrochromosome and gene-rich microchromosome CTs. A transcription microarray analysis of the cells suggests that the radial positioning of CTs impacts only a limited number of genes and plays an active role in repression, rather than in induction. As far as we know, this report is the first observation that an inner nuclear protein is required for the radial distribution of CTs, and will provide new insight into the mechanisms and physical significance of the positioning of CTs in the nucleus. The total RNA was extracted with RNeasy Maxi Kit (QIAGEN) according to the manufacturer protocol. The poly-A mRNA was isolated from total RNA using Oligotex-dT30 Super (JSR corporation) and biotinylated cRNA was synthesized according to Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix). Shortly, 2 ug of poly-A mRNA was reverse transcribed to cDNA using 100 pmol of a T7-Oligo (dT) Primer (Invitrogen), and biotinylated cRNA was prepared by in vitro transcription amplification. For hybridization, 15 ug of biotinylated cRNA was fragmented and added into a hybridization cocktail containing four biotinylated hybridization controls (bioB, bioC, bioD, and cre) as recommended by the manufacturer. GeneChip Chicken genome Arrays (Affymetrix) were hybridized with the cocktail at 45˚C for 16 hours. After washing with Non-Stringent wash buffer (6xSSPE, 0.01% Tween20) and staining with SAPE in the Affymetrix GeneChip Fluidics Station 400, the GeneChips were read using the Affymetrix GeneChip scanner 3000 7G. These systems and data analyses were operated with the GeneChip Operating Software 1.3.
Project description:Male patients (n=6, mean age 62 years) with NYHA III-IV and an left ventricular ejection fraction of <35% despite pharmacological therapy received 35 hours of enhanced external counterpulsation (EECP) over a period of 7 weeks. Before and after treatment, lateral vastus muscle biopsies were obtained and skeletal muscle gene expression was evaluated using the Affymetrix HuGene 1.0 platform. Skeletal muscle gene expression before and after treatment with enhanced external counterpulsation for 7 weeks in 6 male patients with severe heart failure
Project description:Evaluation of the genome wide impact on gene expression of DNA-PK knockdown or enzymatic inhibition. DNA-PK expression is elevated in multiple tumor types. DNA-PK has recently been implicated in transcriptional regulation, so understanding genes modulated by DNA-PK may provide insight into disease progression 6 samples were analyzed. A genome-wide expression array was performed on a GeneChip Human Gene 2.0ST Array (Affymetrix, 902112) with C4-2 cells depleted of DNA-PK or treated with DNA-PK inhibitor for 24 hours
Project description:This array was designed to verify a number of previously identified markers for colo-rectal adenoma. It contains probesets from the Affymetrix Hu133, HuGene and other novel probesets not included on other arrays. 68 samples of colon tissues were analysed, covering the classes normal, adenoma and carcinoma.
Project description:Analysis of global gene expression profiles of FACS-sorted, human Ad5- and CMV-specific CD4 T cells from the same PBMC sample of healthy donros, using affymetrix Human Gene 2.0ST Gene-Chips; The analysis show that Ad5- and CMV-specific CD4 T cells demonstrate differential gene expression profiles even though the cells are from the same PBMC PBMC from healthy donors (n=3) were CFSE-labeled, then equally divided and stimulated with Ad5 and CMV antigens; On day 6 post stimulation, Ag-specific CD4 T cells were sorted by flow based on CFSE-low.
Project description:Comparison of the new generation taxane cabazitaxel with docetaxel in prostate cancer cells Cabazitaxel impacts distint molecular pathways as compared to docetaxel, which could underlie its efficacy after docetaxel treatment has failed in castration resistant prostate cancer patients 12 samples were analysed. A genome-wide expression array was performed on a GeneChip Human Gene 2.0ST Array (Affymetrix, 902112) with C4-2 cells, treated for 16h with 1nM cabazitaxel, docetaxel or vehicle (EtOH), in duplicates. The expression data were RMA normalized, and filtered to remove low-expressing genes. Differential gene expression with corresponding p-values (student’s ttest) was determined of drug-treated over control.
Project description:Comparison of the new generation taxane cabazitaxel with docetaxel in prostate cancer cells Cabazitaxel impacts distint molecular pathways as compared to docetaxel, which could underlie its efficacy after docetaxel treatment has failed in castration resistant prostate cancer patients 12 samples were analysed. A genome-wide expression array was performed on a GeneChip Human Gene 2.0ST Array (Affymetrix, 902112) with LNCaP cells infected with a control plasmid (MSCV-LMP), treated for 16h with 1nM cabazitaxel, docetaxel or vehicle (EtOH), in duplicates. The expression data were RMA normalized, and filtered to remove low-expressing genes. Differential gene expression with corresponding p-values (student’s ttest) was determined of drug-treated over control.