Cell-based phenotypic screening of mast cell degranulation unveils kinetic perturbations of agents targeting phosphorylation

Abstract

Mast cells play an essential role in initiating allergic diseases. The activation of mast cells are controlled by a complicated signal network of reversible phosphorylation, and finding the key regulators involved in this network has been the focus of the pharmaceutical industry. In this work, we used a method named Time-dependent cell responding profile (TCRP) to track the process of mast cell degranulation under various perturbations caused by agents targeting phosphorylation. To test the feasibility of this high-throughput cell-based phenotypic screening method, a variety of biological techniques were used. We further screened 145 inhibitors and clustered them based on the similarities of their TCRPs. Stat3 phosphorylation has been widely reported as a key step in mast cell degranulation. Interestingly, our TCRP results showed that a Stat3 inhibitor JSI124 did not inhibit degranulation like other Stat3 inhibitors, such as Stattic, clearly inhibited degranulation. Regular endpoint assays demonstrated that the distinctive TCRP of JSI124 potentially correlated with the ability to induce apoptosis. Consequently, different agents possibly have disparate functions, which can be conveniently detected by TCRP. From this perspective, our TCRP screening method is reliable and sensitive when it comes to discovering and selecting novel compounds for new drug developments.

Modulation of allergic responses by mitochondrial STAT3 inhibitors

“…Interestingly, Hox et al recently showed that inhibition of STAT3 with C188–9, a STAT3 small molecule, does not affect mast cell degranulation in mouse mast cells. In addition, Qin et al showed that JSI124, a small STAT3 inhibitor which inhibits STAT3 DNA binding and STAT3‐mediated gene transcription, failed to effect mast cell degranulation in RBL cells. Most likely, these results are due to these inhibitors’ weak effect on mitochondrial STAT3, again validating the role of mitochondrial STAT in mast cell degranulation.…”

Effects of Calcium on the Interactions of Acinetobacter baumannii with Human Respiratory Epithelial Cells

“…Compared to traditional endpoint detection, RTCA has the advantages of being non-invasive and highly accurate, as well as providing real-time monitoring, complete TCRPs, and easy operation. It is widely used in cytology research, such as in cell migration and invasion assays, cytotoxicity tests, gene regulation and cell-microenvironment interactions[15,[19][20][21]. Therefore, the obtained TCRPs can provide better information on the effects of calcium on host-bacterial interactions, complementing the results of our microscopy observations.…”

“…Primer sequences for the target gene (ompA, outer membrane protein A) were as follows: 5'-CACAGATAACACTGGTCCACG-3' and 5'-GAATACACGACGGTTCATAGC-3'. The changes in Ab invasion of epithelial cells (including strong adhesion) at different MOIs were anaylsed by comparing the resulting Ct values.Detailed RTCA (real time cellular analysis) experimental procedures have been previously described[15,19,20]. Briefly, 50 μl of medium was added to 16-well E-plates (specific cell culture plates for RTCA) to obtain background readings, which were followed by the addition of 100 μl of a cell suspension (2×105 cells/ml).…”

Effect of Calcium on the Interaction of Acinetobacter baumannii with Human Respiratory Epithelial Cells

“…Finally, bacterialDNA was extracted from 1 × 10 5 cells based on procedures described by Chen et al [26]. qPCR was performed with SYBR Detailed RTCA (real time cellular analysis) experimental procedures have been previously described [15,19,20]. Briefly, 50 μl of medium was added to 16-well E-plates (specific cell culture plates for RTCA) to obtain background readings, which were followed by the addition of 100μl of a cell suspension (2×10 5 cells/ml).…”