Project description:CTCF, H2AFZ and FOXA1 genomic recruitment sites were determined using ChIP-chip while MeDIP-chip was used to monitor DNA methylation levels. Amplified and labeled DNA was hybridized to Affymetrix tiling arrays covering human chromosomes 8, 11 and 12. Cells used in this study are: MCF7 breast cancer cells, LNCaP prostate cancer cells, MDA-MB-231 breast cancer cells stably transfected with a FOXA1 expression vector (MDA231-FOXA1) or the empty control plasmid (MDA231-CTRL). H3K4me2 genomic distribution was determined using ChIP-chip. Amplified and labeled DNA was hybridized to Affymetrix tiling arrays covering human chromosomes 8, 11 and 12. Cells used in this study are MDA-MB-231 breast cancer cells stably transfected with a FOXA1 expression vector (MDA231-FOXA1) or the empty control plasmid (MDA231-CTRL).
Project description:Aerobic glycolysis is a hallmark of cancer glucose metabolism. Here we suggest that extracellular vesicles (EVs) originating from cancer cells can modulate glucose metabolism in the recipient cancer cells and induce cell proliferation and aggressive cancer phenotypes. Two breast cancer cell lines with different levels of glycolytic activity, MDA-MB-231 and MCF7, were selected and co-cultured, as the originating and recipient cells. The change in 18F-fluorodeoxyglucose (FDG) uptake of the recipient MCF7 cells was assessed after co-culture with the MDA-MB-231 cells. Proteomics analysis was performed to investigate the changes in the protein expression patterns in the recipient MCF7 cells. FDG uptake by the recipient MCF7 cells was sig-nificantly increased after co-culture with the MDA-MB-231 cells.
Project description:Epithelial-to-mesenchymal transition (EMT) is a fundamental process in development and disease. If aberrantly activated it is a trigger for tumour progression and metastasis (Thiery et al 2009 Cell). It is now known that EMT activation is also associated with the maintenance of stem-cell properties (Mani et al. 2008 Cell). Since Zinc-finger enhancer binding transcription factor 1 (ZEB1) is a crucial EMT activator, we analyzed the changes in the gene expression profile that accompany shRNA mediated loss of ZEB1 in MDA MB 231 basal type breast cancer cells. MDA MB 231 is a cell line that exhibits mesenchymal characteristics, but reverts to an epithelial phenotype upon ZEB1 knock down (Spaderna et al. 2008 Cancer Research). MDA MB 231 cells were stably transfected with control (GFP) or ZEB1 shRNA. Upon puromycin selection, single cell clones were picked and characterized. Cells from two control versus two ZEB1 knockdown clones were harvested, total RNA was isolated and processed to hybridization.
Project description:MCF7 and MDA-MB-231 breast cancer cell lines were cultured in DMEM-F12 containing 10% FBS (Lonza) 100U/ml penicillin and 100 µg/ml streptomycin (Lonza). Transfecting third generation packaging vectors using Poly-ethylenimine into HEK293T cells generated lentiviral particles (17). MCF7 and MDA-MB-231 cells were stably transduced with lentivirus containing pINDUCER20-FOXO3.A3, allowing doxycycline induced expression of constitutively active FOXO3 (FOXO3.A3). Cells were treated with 20% FBS or 10 µM PI3K inhibitor LY294002 (Selleckchem) for 16 hours to activate and inactivate the endogenous PI3K pathway, respectively. FOXO3.A3 expression was induced by 16 hours treatment with 10 ng/ml doxycycline.
Project description:RNA was isolated from ectopically sFRP1-expressing MDA-MB-231 cells and control MDA-MB-231 cells and as well from tumor lysates arising from these cells as nude mouse xenograft. Gene expression profiles for these samples were investigated using Affymetrix arrays. Experiment Overall Design: MDA-MB-231 human breast cancer cells were stably transfected with human sFRP1 encoding vector or empty vector as control. After the selection with antibiotics, three clones of MDA-MB-231/sFRP1 and three clones of MDA-MB-231/control were selected. These six clones were cultured individually in DMEM 10% FCS with 1mg/ml G-418. When cells reached 70-80% confluence, RNA was isolated from the cells. In parallel, the three clones of MDA-MB-231/sFRP1 and the three clones of MDA-MB-231/control were pooled respectively. One million of cells from each pool were suspended in 100ul PBS and injected to fat pads of female balb/c nude mice (6 mice were injected with MDA-MB-231/sFRP1 and 5 mice were injected with MDA-MB-231/control) to do a xenograft experiment. A few - several weeks after, mice were sacrificed when tumor reached a certain size, tumors were taken and RNA was isolated using trizol reagent.
Project description:Gene expression analysis of MDA-MB-231 breast cancer cells cultured in suspension {and stably expressing control shRNA, shPHLPP2 or GFP-Akt DD (GFP-HA-Akt-T308D S473D)}. Results provide insights into regulation of molecular pathways by PHLPP2 and Akt signaling in matrix deprived condition.
Project description:To interrogate the hypoxia response pathways affected by the depletion of CAIX in BC cells, we performed gene expression profiling using SurePrint G3 Human Gene Expression 60K microarrays in MDA-MB-231-shCAIX, MCF7-shCAIX and the respective control cells grown as multicellular spheroids under hypoxic or normoxic conditions. The human breast cancer cell lines MDA-MB-231 and MCF7 were purchased from the European Collection of Cell Cultures (ECACC, UK) and maintained as recommended by the manufacturer. To generate stable CAIX silenced cells, cell lines were transfected with a pool of three plasmids each encoding CAIX-specific shRNA (1 µg) and a negative control (NC) plasmids encoding scrambled shRNAs (1 µg) (Santa Cruz Biotechnology, TX, USA) using shRNA transfection medium and shRNA transfection reagent (Santa Cruz Biotechnology, USA) according to the manufacturers’ protocol. For selection of stably transfected cells, puromycin dihydrochloride (Santa Cruz Biotechnology, USA) was added to medium 48 h post-transfection (6 µg/ml for MCF7, 2 µg/ml for MDA-MB-231 cells). The transfected cells were plated at density 2e+3 cells per ml of serum-free DMEM/F12 medium supplemented with EGF (20ng/ml, R&D Systems, #236-EG-200), bFGF (10ng/ml, SantaCruz, #sc-4573), hydrocortisone (50 ng/ml, Sigma-Aldrich, #H0135-1MG) and 1B27 (Invitrogen, #17504001) and grown as multicellular spheroids for 5 days. To establish hypoxic conditions, the cells were cultured at 1% oxygen, 94% N2 and 5% CO2 at 37oC using humidified multi-gas incubator (Sanyo, Sanyo Electric Co.,Ltd.) for 48 hours. The normoxic control cells were incubated at 37oC with 5% CO2 in a humidified incubator (Panasonic, Panasonic Healthcare Co., Ltd.). The efficiency of CAIX silencing was assessed by qRT-PCR and immunofluorescence before the experiments.
Project description:Using microarray, we compared miRNAs expression profiles of MDA-MB-231 cells transfected with myocardin and empty vector (pcDNA3.1) and found that 25 miRNAs were significantly changed in myocardin-transfected groups (17 up-regulated and 9 down-regulated miRNAs). Moreover, we showed that 18 of 25 miRNAs significantly regulated by myocardin were inhibited by ERα in MDA-MB-231 cells. In addition,through a microarray approach, we identify the subset of miRNAs modulated by ERα in MDA-MB-231 cells. Our results determined that ERα may function as tumor-promoter through down-regulating expression of 3 miRNAs (miR-26b, miR-146a and miR-331-3p) in MDA-MB-231 cells.
Project description:Analysis of breast cancer MDA-MB-231 cells stably over-expressing SUV420H2, a histone H4K20 methyltransferase. Several genes were significantly up- or down-regulated. Results provide insight into the molecular mechanism by which H4K20me3 contributes to gene expression. SUV420H2 stably over-expressing MDA-MB-231 cells were cloned. Then total RNA was extracted from the SUV420H2 over-expressing cells and the parental MDA-MB-231 cells.
Project description:Analysis of breast cancer MDA-MB-231 cells stably over-expressing SUV420H2, a histone H4K20 methyltransferase. Several genes were significantly up- or down-regulated. Results provide insight into the molecular mechanism by which H4K20me3 contributes to gene expression. SUV420H2 stably over-expressing MDA-MB-231 cells were cloned. Then total RNA was extracted from the SUV420H2 over-expressing cells and the parental MDA-MB-231 cells.