Project description:Results of growing MCF10A cells continuously in serum free media supplemented with EGF (MCF10A) or AREG (MCF10A+AREG) followed by 24 hours of ligand withdrawl and measuring gene expression provides information as to what genes are regulated by AREG and EGF in a normal mammary epithelial cell model MCF10A cells continuously in serum free media supplemented with 10ng/ml of EGF (MCF10A) or 20ng/ml of AREG (MCF10A+AREG) followed by 24 hours of ligand withdrawl. Total RNA was collected and genome-wide analysis of expression was performed on RNA from each cell line.
Project description:Results of growing MCF10A cells continuously in serum free media supplemented with EGF (MCF10A) or AREG (MCF10A+AREG) followed by 24 hours of ligand withdrawl and measuring gene expression provides information as to what genes are regulated by AREG and EGF in a normal mammary epithelial cell model
Project description:The presence of extra centrosomes is a feature of human tumours. However, it is unclear what are the changes elicited by the presence of these abnormalities. To determine the changes in gene expression induced by centrosome amplification, human mammary epithelial cells, MCF10A, expressing a doxycycline inducible PLK4 cDNA were used. Centrosome amplification was induced for 48 hrs upon the addition of doxycyclin to the culture medium. RNA was purified from MCF10A cells without extra centrosomes (no dox) and with extra centrosomes (dox) and processed for microarray analysis.
Project description:We analyzed the effect of tetracycline (Tet)-inducible amphiregulin (AREG) silencing on the transcriptome of immortalized human N/TERT keratinocytes in the presence or absence of exogenous rhEGF. Tetracycline-mediated AREG silencing significantly altered the expression of 2,331 genes, 623 of which were not normalized towards normal by treatment with EGF. Genes irreversibly up-regulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. A significant proportion of the irreversibly down-regulated genes featured upstream binding sites recognized by FoxM1
Project description:Mammary epithelial cells MCF10A and HER2 overexpressing MCF10A cells were grown on matrigel in the absence or presence of epidermal growth factor. Cells were lysed and RNA was collected at 1.5,3,5,7,9 days. Total 20 arrays from four time course experiments. MCF10A and MCF10A-HER2 (overexpressed HER2) were plated on Matrigel in the absence or presence of 20 ng/ml of EGF. This design creates four panels of experiments of 5 time points each.
Project description:MCF10A cells derived from spontaneously immortalized normal human mammary epithelia were subjected to EGF/SERUM stimulation for 0,20,40,60,120,240 and 480 minutes. We used microarrays to understand the temporal regulation of the cellular EGFR cascade. Experiment Overall Design: MCF10A cells were grown in DME:F12 medium supplemented with antibiotics, [10 mg/ml insulin, 0.1 mg/ml cholera toxin, 0.5 mg/ml hydrocortisone, heat-inactivated horse serum (5%); defined as 'serum' in the text] and 10ng/ml EGF. Cells were serum deprived for 24 hours. Following stimulation with EGF/SERUM for 0,20,40,60,120,240 and 480 minutes.
Project description:The epidermis, the most superficial layer of human skin, serves a critical barrier function, protecting the body from external pathogens and allergens. Dysregulation in the epidermal differentiation process contributes to barrier dysfunction and is implicated in the pathology of various dermatological diseases, including atopic dermatitis (AD). Mucopolysaccharide polysulfate (MPS) is used as a moisturizing agent for xerosis in AD patients. However, its mechanism of action on keratinocytes, the main constituent of the epidermis, remains unclear. In this study, we investigated the impact of MPS on keratinocytes by subjecting adult human epidermal keratinocyte (HEKa) cells and three-dimensional cultured keratinocytes to MPS treatment, followed by transcriptome analysis. The analysis revealed that MPS treatment enhances keratinocyte differentiation and suppresses proliferation. We focused on amphiregulin (AREG), a membrane protein that belongs to the epidermal growth factor (EGF) family and possesses a heparin-binding domain, as a significant target of the MPS among the genes altered by MPS. It is revealed that MPS exerts an inhibitory effect directly on AREG, rather than on the EGF receptor or other members of the EGF family. Furthermore, it is suggested that AREG leads to a reduction in epidermal barrier function, whereas MPS contributes to barrier enhancement through AREG inhibition. Collectively, these findings suggest that MPS modulates barrier function through the inhibition of AREG, offering insights into potential therapeutic strategies for skin barrier restoration.
Project description:Background: The gonadotropin-induced resumption of oocyte meiosis in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. Both the gonadotropins and the EGF-like peptides possess the capacity to stimulate resumption of oocyte meiosis in vitro via activation of a broad signaling network in cumulus cells. To better understand the rapid genomic actions of gonadotropins (FSH) and EGF-like peptides, we analyzed transcriptomes of cumulus cells at 3 h after their stimulation. Methods: We hybridized aRNA from cumulus cells to a pig oligonucleotide microarray and compared the transcriptomes of FSH- and AREG/EREG-stimulated cumulus cells with untreated control cells and vice versa. The identified over- and underexpressed genes were subjected to functional genomic analysis according to their molecular and cellular functions. The expression pattern of 50 selected genes with a known or potential function in ovarian development was verified by real-time qRT-PCR. Results: Both FSH and AREG/EREG increased the expression of genes associated with regulation of cell proliferation, cell migration, blood coagulation and extracellular matrix remodeling. FSH alone induced the expression of genes involved in inflammatory response and in the response to reactive oxygen species. Moreover, FSH stimulated the expression of genes closely related to some ovulatory events either exclusively or significantly more than AREG/EREG (AREG, ADAMTS1, HAS2, TNFAIP6, PLAUR, PLAT, and HSD17B7). In contrast to AREG/EREG, FSH also increased the expression of genes coding for key transcription factors (CEBPB, FOS, ID1/3, and NR5A2), which may contribute to the differing expression profiles of FSH- and AREG/EREG-treated cumulus cells. Conclusions: The impact of FSH on cumulus cell gene transcription was higher than the impact of EGF-like factors in terms of the number of cell functions affected as well as the number of over- and underexpressed genes. Both FSH and EGF-like factors overexpressed genes involved in the post-ovulatory switch in steroidogenesis and tissue remodelling. However, FSH was remarkably more efficient in the up-regulation of several specific genes essential for ovulation of matured oocytes and also genes that been reported to play an important role in maturation of cumulus-enclosed oocytes in vitro.
Project description:Neuronal differentiation of PC12 cells in response to NGF is a prototypical model in which signal duration determines a biological response. Sustained ERK activity induced by NGF, as compared to transient activity induced by EGF, is critical to the differentiation of these cells. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2-4 hrs, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially upregulated in response to NGF. PC12 cells that were starved in low serum media for 24 hrs were treated with NGF (50ng/mL) or EGF (25ng/mL) for 2 or 4 hrs, or left untreated. Total RNA for 3 independent biological replicates was extracted and subjected to Affymetrix Rat Gene 1.0ST Arrays. The 69 genes that were preferentially upregulated by NGF compared to EGF met the following criteria: NGF/No treatment log2> 1, FDR p value< 0.01 and NGF/EGF log2> 0.75, FDR p value< 0.01.
