The work I’ve been doing with Dr. Nancy Krucher involves the very invasive disease, Pancreatic cancer. We have worked on a possible solution to the invasive behavior of cancer cells that comprises of knocking down a specific phosphatase subunit and its effect on the Retinoblastoma tumor suppressor protein. We also studied the effects of the drug Erlotinib on cell death. At the start of the year, I worked with 96-well plates for various experiments. Doing this allowed my pipetting skills to improve greatly and I gained more knowledge of knockdowns and cell growth. Some experiments included creating and testing several concentrations of Erlotinib and finding it’s optimal concentration for cell death. Then after finding this concentration, we treated the MiaPaCa-2 pancreatic cancer cells with PNUTS knockdown to inhibit invasion for 48 hours and proceeded with Erlotinib for 24 hours. Afterwards, to count cells we used Cell Titer-Glo spectrophotometer assays to see how effective the combined treatment was in killing off cells. This process took several experiments for me to get the hang of the techniques but the results were great. There were plenty of experiments that were flops, and some that were good but had very big error bars for our data (which isn’t so great). However, multiple experiments of mine showed a decrease in cell count when the cells were treated with both PNUTS knockdown and Erlotinib.
To further look into how PNUTS knockdown would affect the cells, we studied its effects on invasion. I had to perform immunoblotting experiments to see the differences in expression for several EMT (Epithelial-Mesenchymal Transition) markers. Before this experiment, I didn’t know a thing about EMT and it honestly helped me so much in one of my classes afterwards when reviewing other scientific papers. Our main focuses were the markers Zeb, N-cadherin, E-cadherin, and Vimentin. This took me so long to get used to. I had to learn how to lyse cells and extract protein and create samples to run a gel with. The hardest part was finding the concentrations of protein and creating samples properly. If this wasn’t done correctly then my gels were pointless and the bands on my nitrocellulose paper wasn’t reliable. I was able to finally work through my mistakes with some practice and obtained some good results. I found a decrease in both Zeb and N-cadherin expression; which signifies a reduction in invasiveness due to the knockdown. I’m still currently working on experiments like what was just mentioned but with other markers and even touching on different pathways affecting invasion as well.
Throughout this whole experience I’ve realized how great it is to work in the lab and conduct such interesting research. My goal in life is to become a doctor but with my experiences this past year I actually am looking into joining a program to obtain an MD-PHD degree. I want to continue researching in the future. Yes, it gets frustrating when experiments don’t go as planned or when results get messed up, but as Dr. Krucher would always remind me, everything takes time and practice. It is always a learning experience. Even when I thought I knew how to do everything properly there was always something else to be learned and practiced. I am so proud and happy with what I’ve done in this past year and so thankful I’ve been able to do it. I’m very appreciative of the UGR program and Dr. Krucher (for putting up with all my mistakes!). Her guidance has helped me greatly and I hope the project continues to bring positive results!