Blog Post#1: Induction of PNUTS Knockdown in 3-D cultures of acinar-structured breast cells leads to dephosphorylated Rb

The proposed title of my research is as follows: “Induction of PNUTS Knockdown in 3-D cultures of acinar-structured breast cells leads to dephosphorylated Rb.” The purpose of my project is to observe the growth of breast cells in a 3-D environment as well as introduce a protein called PNUTS into the culture to examine if the expected phosphorylation of Rb and apoptosis occurs. I have some research providing support for apoptosis through induction of PNUTS siRNA into two different breast cell lines (MCF7’s & Hs578T’s) causing activation of Rb-directed PP1 activity thus leading to dephosphorylated Rb and finally apoptosis all in a 2-D environment. Therefore, I am expanding on these results and shifting from a 2-D environment to a 3-D one and focusing on if Rb can indeed be dephosphorylated and if apoptosis will continue to occur through these similar procedures.

Due to the fact that conducting in research takes a large sum of time to develop skills for and to gain an abundance of knowledge, my goals are to become completely familiar and comfortable with all of the necessary techniques for my project as well as develop a larger collection of facts and information on breast cancer research in general; hopefully I am able to accomplish these two goals by the end of this semester. Additionally, I hope to obtain a lot of useful and credible results not only for myself but also for my mentor, Dr. Nancy Krucher, to utilize for her own work. A few objectives I have for my project are to maintain a collection of healthy MCF7 cells as they grow in their cultures, to completely understand the purpose and technique of the western blot within the next week or two, and to oversee and carry out the full experimental design which roughly includes culturing, addition of PNUTS, and the western blot, all within the next few weeks. Furthermore, I hope to gain a positive experience and stronger skill set in the methods of research in addition to growing a deeper passion and acceptance for research and its many unique factors and procedures. I hope to become widely knowledgeable on my topic in addition to be able to effectively decipher other researchers’ results, journals, articles, etc to help me make use and create a better understanding of my own compiled work. Finally, I hope to achieve very substantial and successful results from my project in order to progress further and continue to conduct in research with Dr. Krucher after the conclusion of this specific project.

A few of the experimental methods I will be engaging in are cell culturing, plating and feeding cells, protein extraction in 3-D’s, cell lysis, and protein assays, administration of the PNUTS siRNA protein sequence, and the western blot. I must practice the art of cell culturing in a 3-D environment which is new not only to myself but to many other researchers as well due to the fact that working in 3-D cultures and utilizing 3-D models is a somewhat brand new concept being expanded on in the field of research. The MCF7 cells I am working with are plated in small, plastic, 8 chamber slides fixated with a laminin and collagen matrix called Matrigel. Cells must be fed every four days in order to grow and form clusters that resemble acini like structures which are the true arrangements that are found in the ducts in breasts. Protein extraction will take place in order remove the cells from the Matrigel by dissolving the matrix and forming a pellet of the cells through centrifugation in a microcentrifuge tube. Through cell lysis procedures and protein assays, I will be able to determine the amount of protein in my sample. Once I introduce PNUTS into my experiment, I will be able to perform gel electrophoresis and western blotting techniques in order to examine the protein sequences present in my sample, and more importantly, to center my attention on the activity of the Rb protein.

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