Optimization of Nucleo-Cytosol Separation to capture EZH2 subcellular localization in Non-Small Cell Lung Cancers

Abstract

EZH2 is a protein subunit known for its role in transcriptional expression and repression. Recent studies have detected EZH2 to be overexpressed in prostate, lung, and breast cancer and therefore is thought to have a part in decreasing drug efficiency. Since its presence is over-detected in these cancers, it is intrinsic to identify the location of EZH2 and whether it is present in the nucleus or cytosol to understand its role in lung carcinogenesis and develop targeted treatments. In this study, three commercially available lung cancer cell lines H2228, H522, and A427 were used to optimize the nucleo-cytosol separation technique. Aggregates of 2 million, 4 million, and 6 million cells were tested to establish the optimal input material for the separation. From each condition, the nucleus and cytosol were extracted using a commercially available kit (NE-Per Nuclear and Cytoplasmic Extraction Reagents from Thermo Scientific) and Western Blot analysis was used to determine the level of purity of each cellular compartment using a cytosolic (HSP90) and a nucleus (PARP) marker. Different washing protocols were also tested to assure purity of each compartment. Based on previous  trials, an optimal protocol needs to be established for each cell line. For example, it was determined that the A427 cell line required 6 million cells with one wash during the nucleo-cytosol separation for optimal separation of the two cellular compartments while the H 2228 cell line showed the clearest results when an aggregate of 2 million cells was washed once during the nucleo-cytosol separation. Conservely, the western blot of the H522 cell line was non-compliant with any of the detailed aggregate amounts whether or not washed. Further trials need to be conducted with the H522 cell line using a higher aggregate of cells in order to view clear results. Nonetheless, this technique is critical to discovering the presence of EZH2 and phosphorylated EZH2 in the nucleus and cytosol, playing  an important role in determining how EZH2 interferes in cancer treatment.