Core clock regulators in dexamethasone-treated HEK 293T cells at 4 h intervals

Abstract

Objective The study of the circadian clock and its mechanisms is easily facilitated through clock resetting in cell culture. Among the various established synchronizers of the circadian clock in cell culture (temperature, serum shock, glucocorticoids), the artificial glucocorticoid Dexamethasone (DEX) is the most widely used. DEX treatment as a protocol to reset the circadian clock in culture gives simple readout with minimal laboratory requirements. Even though there are many studies regarding clock resetting in culture using DEX, reference points or expression patterns of core clock genes and their protein products are scarce and sometimes contradict other works with similar methodology. We synchronise a cell line of human origin with DEX to be used for studies on circadian rhythms. Results We treat HEK 293T cells with DEX and describe the patterns of mRNA and proteins of core clock regulators, while making a clear point on how CLOCK is less than an ideal molecule to help monitor rhythms in this cell line.

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2

“…In this context, we tested whether the clock protein PER2 in HEK-293T cells can be oxidized by S-nitrosation, thereby leading to changes in the stability of its monomer. HEK-293T cells were selected due to their extensive use as an established model for studying the circadian molecular clock regulation (e.g., resetting with dexamethasone [ 10 ], peroxide control of CLOCK cysteine oxidation on CLOCK-BMAL1 heterodimer activity [ 8 ], or its outputs (e.g., proteasome activity on oxidized proteins [ 11 ], enzymatic function in oxidative stress [ 12 ]). Our results show that PER2 exhibits cysteines susceptible to oxidation and that treatment with the S-nitrosating compound S-nitroso-L-cysteine (CysNO), or with hydrogen peroxide, reduced its monomer, subsequently increasing homodimers and oligomers in a time- and dose-dependent manner.…”