4 oh ppa

Real-time bioluminescence recording was conducted according to a previously described method with minor modification (Lee et al., 2016 (link)). A day before the real-time recording, MEF cells were seeded in 35 mm dish (Fisher Scientific, USA) at 50% confluence. After 24 h, culture media was changed to synchronization media with 200 nM dexamethasone (Sigma-Aldrich, USA) to synchronize circadian rhythms of cell population (So et al., 2009 (link)) for 2 h. To record the real-time bioluminescence, synchronized cells were cultured in recording media with 200 μM D-luciferin (Promega, USA) and culture dishes were sealed with parafilm (Sigma-Aldrich). Next, the dishes were placed in Kronos, a real-time bioluminescence recording device (ATTO, Japan), at 37°C and 5% CO₂, and luciferase (Luc) activity in each dish was measured for 1 min every 10 min for 3 to 5 days. For the dose-response effect of 4-OH-PPA and PPA, the cells were treated with four different doses, 0.125 mM, 0.5 mM, 1 mM, and 2 mM of 4-OH-PPA (Sigma-Aldrich) and PPA (Sigma-Aldrich) using two different administration modes, either immediately after the nadir or the peak of the oscillation (Fig. 1B). To verify the effect of 4-OH-PPA and PPA, their precursors, tyrosine (Sigma-Aldrich) and phenylalanine (Sigma-Aldrich), were examined initially as the control experiments.