UNIVERSITY OF LOUISVILLE
Grant: $64,898 - National Institutes of Health - Aug. 4, 2009
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Award Description: Summary narrative of ARRA funded project activity/milestones: Research design and methods and data analysis for the summer research supplement: 1. General Research plan: The summer hs student, undergraduate student, and hs science teacher worked with PI and members of her laboratory including a research associate, a postdoctoral fellow and a technician. The general plan follows research currently underway in our lab and will be accomplished over the next 2 years of the grant period. 1. The hs student will learn basic lab skills including buffer and gel-making, autoclaving solutions, running western blots, scanning films, and learning data-quantitation. 2. The activities in which the undergraduate student and hs science teacher will participate include cell culture for the growing of cells and their treatment, RNA isolation, protein isolation, quantitation of protein and RNA, determining RNA quality, reverse transcription, quantitative real-time PCR (Q-PCR), and western blot. 3. MCF-7 human breast cancer cells will be grown in phenol red-free IMEM and ‘serum starved’ in 5% DCC-treated FBS prior to hormone treatment. Treatments include 1 and 10 nM E2, 100 nM 4-OHT, 1 uM ICI 182,7780, 10 nM PPT (selective ER?-agonist) or 10 nM DPN (selective ER? agonist). Whole cell or cytosolic versus nuclear extracts (WCE, CE, and NE) will be used for Coimmunoprecipitation assays with NRF-1, ER? and ER? antibodies. Western blots will be used to examine interactions. 4. GST-expression vector constructs with NRF-1 and cyclin D1 genes will be created for direct protein: protein interaction assays (GST-pulldown assays with GST-NRF-1 and GST-cyclin D1- we already have GST-ER?). We plan to involve the hs teacher in cloning efforts. 5. GST pulldown assays will be performed to examine whether there is direct or indirect interaction between NRF-1, cyclin D1, ER? and ER? antibodies will be used to examine the levels of proteins immunocaptured in the GST-pulldown experiment. 6. Q-PCR will be used to evaluate the effect of either overexpressing or knocking down (siRNA) NRF-1 on cyclin D1 transcription in MCF-7 breast cancer cells. This would involve the undergraduate student in learning cell culture for the growing of cells and their treatment, RNA isolation, RNA quality assessment and quantitation, reverse transcription (RT) and quantitative Q-PCR. 7. To determine how E2 and 4-OHT impact the direct interaction of ER? and ER? with the NRF-1 promoter and whether different coregulators are recruited in a ligand- and cell- specific manner, chromatin immunoprecipitation (ChIP) assays will be performed. Again, these assays will involve cell treatments that can be performed by the undergraduate student and hs science teacher. 8. The effect of siNRF-1 on E2-regulated cell cycle and apoptotic genes, proteins, and cell cycle will be examined. FACS will be used to examine cell cycle and apoptosis. If siNRF-1 increases apoptosis, PARP, caspase-3,and capase-9 cleavage will be examined.
Project Description: Summer positions were hired and they completed the research activities as stated in the award description above.
Jobs Summary: 3 New jobs for 1 summer.Funds were requested for hiring 1 high school science teacher, 1 high school student, and 1 undergraduate student to work on this project in a research lab for 1 summer. (Total jobs reported: 3)
Project Status: Less Than 50% Completed
This award's data was last updated on Aug. 4, 2009. Help expand these official descriptions using the wiki below.
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