(1138-C) Quantitative Analysis of Protein Translocation using High-Content Imaging and YT-SOFTWARE

Abstract: Protein translocation between intracellular compartments is a fundamental process for the maintenance of cellular functions like the trafficking of transcription factors from the cytoplasm to the nucleus after activation by external or internal stimuli. Consequently, the development of easy and fast methods to visualize and quantify protein trafficking is important in various scientific fields. A conventional approach for analyzing the localization of proteins in the cell is immunofluorescence (IF) staining. However, performing IF stainings presents challenges, due to complex protocols demanding a considerable amount of hands-on time. Moreover, imaging can be time-intensive and image analysis often requires additional software. To address these challenges and develop an assay suitable for automation, we performed IF stainings directly in multiwell plates imaged them with our high-content imager CELLAVISTA 4K and implemented a new image analysis tool in our YT-SOFTWARE.
To do so, we carried out translocation assays of two commonly known transcription factors moving between the cytosol and nucleus, signal transducer and activator of transcription 3 (STAT3) and nuclear factor k-light-chain-enhancer of activated B cells (NF-ĸB). Trafficking was induced by treating A549 and HT29 cancer cells with the cytokines Interleukin-6/Interleukin-6 receptor α (IL-6/IL-6Rα) or tumor necrosis factor-alpha (TNF-α) over time. Afterward, IF stainings were performed using antibodies, CellMask Deep Red to segment the cells, and Hoechst 33342 to stain the nuclei. The plates were automatically imaged with CELLAVISTA 4K and analyzed with the new image analysis application Translocation (1F) of the built-in YT-SOFTWARE. The application automatically analyzed the average fluorescence intensity in the nucleus and cytoplasm and calculated a nucleus/cytoplasmic translocation ratio. Using these parameters, an increase of STAT3 and NF-ĸB fluorescence signal in the nucleus compared to the cytosolic region was observed after treatment.
Altogether, we developed an easy-to-use tool to analyze protein translocation based on IF stainings in automated high-throughput settings. Imaging and image analysis are performed within the same software platform, reducing costs, and allowing the analysis to be started on-the-fly during imaging, which further increases the overall throughput.