Exploring redox-associated single-cell variability with imaging-based spatial proteomics

Sammanfattning

Many have heard that antioxidants play a key role in living a long and healthy life, but not all know the reason for it. The importance of antioxidants lie in their ability to lower the concentration of reactive oxygen species (ROS), such as hydrogen peroxide, specifically when an imbalance of redox state causes oxidative stress. ROS are known to be a contributing factor in multiple diseases including cancer, which makes the study of their impact on cells and proteins very valuable for the understanding of the causes and potential treatments of those diseases. This study focuses on the impact of hydrogen peroxide on eleven redox-associated proteins. These proteins are: ACAD9, ECHS1, ETFB, HADHA, L2HGDH, CAT, HSD17B4, TYR, ALDH6A, CtBP1, and GAPDH. Specifically, we explored how their expression levels change due to hydrogen peroxide-induced oxidative stress as well as if there is a change in the singlecell variability. We conducted experiments experiments on the cells from the U2OS line, using 96-well plates. The experimental setup included three different conditions of incubation time and six different concentrations of H2O2. We performed immunostaining of the targeted proteins, cell structures and with the addition of a fluorescent probe for ROS, took images of each well and used in a Python script to retrieve the fluorescence data of the cells with and without H2O2 treatment. When the data was collected and analyzed, the results from the plates incubated for 1.5h and 24h, were disregarded due to their unreliability. Upon analyzing, many proteins showed different tendencies of fluorescence for different concentrations of H2O2 added. For some, the mean protein fluorescence increased with the added H2O2 concentration, while others indicate a slight decrease of fluorescence or no clear difference. With the collected results, we hope to contribute to the mapping of protein expressions in cancerous cells during a state of oxidative stress. As for the single-cell variability, it provides an important aspect to consider during cancer treatment, since the variability