Results from Trial 1/ Starting Trial 2

At the beginning of this week, I looked at my Trial 1 slides for no treatment/ chloroquine that I made the week before. I examined the twelve chloroquine slides and took pictures of those slides. I need to perform statistics on the slides but I will do that throughout this week. In general, the majority of the cells on the chloroquine slides were not viable and the cells that were alive were scarce. One explanation for this could have been forceful washing during the cell fixation process. This week when I do cell fixation, I will make sure to be careful when washing the cells using the autopipette. I have also done cell culture for Trial 2 of no treatment/chloroquine. I will add my DNA conditions to my cells today and add the autophagy inhibitor drug, chloroquine, to the cells tomorrow.

Starting my independent project

This week, I have started my first trial of my independent project. As a reminder, I am trying to determine if pseudophosphatase, MK-STYX is reducing stress granule formation via the autophagy (cell recycling pathway). To tackle this question, I have to culture mammalian cells, transfect the cells with the different DNA plasmid conditions,  induce and inhibit the autophagy pathway via pharmaceutical drugs, fix the cells, and examine the cells under the fluorescence microscope.  This week I am starting my first trial of my experiment looking at the results of added no drug and the autophagy inhibitor drug. Thus far, I have cultured and transfected mammalian cells with my different DNA plasmid conditions. Later in the week, I will add autophagy inhibitor drug, fix the cells, and examine the cells using fluorescence microscopy. I am excited to see preliminary results of this experiment.

Pseudo-protein may reduce cell trauma via non-specific cell recycling

Just like us, cells get stressed out. Cells become stressed when there is an external force like ultraviolet light, heat, or harsh chemicals that cause cellular conditions to be imbalanced. To cope with the external stressors, cells form these dense aggregations in the cytoplasm composed of messenger RNA (mRNA) and proteins. These dense aggregations are called stress granules. Within these stress granules, mRNAs are sorted, proteins are modified, and proteins targeting the stress response are translated. Stress granules allow cells to continue to “run” or operate under stressful conditions but stress granules are meant to be temporary bodies. If stress granules are retained within the cell, they can cause complications in the cell which could lead to common diseases like cancers and neuro-degenerative diseases like Alzheimer’s. In order to prevent diseases, the cell has to actively get rid of stress granules when the external stressors cease. Research shows that a specific pseudo-protein called MK-STYX reduces stress granules in cells but the method of how MK-STYX reduces stress granules is still unknown. Research also shows that a non-specific degradation pathway called the autophagy pathway clears stress granules from cells. Knowing that both MK-STYX and autophagy reduce stress granules in cells, I am trying to determine if MK-STYX is reducing stress granules via the autophagy pathway. During the summer, I will culture and transfect HeLa G3BP-GFP and HEK G3BP-GFP cells with MK-STYX. I will then add either an autophagy inducer drug or an autophagy inhibitor drug to the cells and induce stress granules formation by over-expressing G3BP-1 (a nucleator protein and stress-granules forming protein). Once all of these aspects are integrated in the cells,  I will examine the cells using fluorescence microscopy and will use quantitative analysis to compare cells of differing conditions.