Project description:DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by the characterisation of select model genes, the abundance and distribution of such structures in the human genome have remained unclear. Here we utilise high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified i-motif structures that are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases.
Project description:MCF7 cells were infected with retrovirus to overexpress wild-type and mutant miR-222 Measure miRNA expression level in MCF7 cells after overexpressing wild-type and mutant miR-222
Project description:The epithelial to mesenchymal transition (EMT) is implicated in the metastatic spread of breast cancer cells. EMT transcription factors (TF) regulate different stages of EMT states. In breast cancers, estrogen receptor α (ERα) maintains the epithelial characteristics of breast tumors and is indispensable for efficient endocrine therapy. In this study we investigate whether and how ZEB1, an EMT-TF affects ERα signaling at early stages of EMT and metastasis. We did ERα ChIP-seq in wild type MCF7-V cells for comparative studies. We also did ERα ChIP-seq in cells stably expressing a doxycycline-inducible construct to express ZEB1. This was to determine the impact of ZEB1 on the ERα cistrome in MCF7-V breast cancer cells induced with DMSO, 17-beta estradiol (E2), and forskolin + 3-isobutyl-1-methylxanthine (IBMX) (FI).
Project description:Wild type (wt) MCF7 cells, modelling breast cancer at primary diagnosis, were cultured in phenol red-free RPMI supplemented with 10% FBS and 1nM estradiol (E2). Long-term oestrogen deprived (LTED) cell lines, which model resistance to endocrine therapy, were cultured in phenol red-free RPMI in the absence of exogenous E2 and supplemented with 10% dextran charcoal-stripped bovine serum (DCC). Samples were harvested at baseline and at the point of resistance (LTED). In order to do comparative analysis in the ER-interactome of wt-MCF7 and MCF7-LTED cells, ER-RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) was conducted in these cells.