Screening Circadian Period Modulators

Screening of small molecules changing circadian period was performed as previously described (Uehara et�al. 2019) (link), using an ITbM chemical library (Ziadi et�al. 2017 , Toh et�al. 2018 (link)), in which all molecules were dissolved in dimethyl sulfoxide (DMSO, Molecular biology grade, Nacalai, Japan), CCA1:LUC transgenic seedlings and an automated luminescence monitoring system (CL96, Churitsu). Molecules dissolved in DMSO at 1 mM were diluted with half strength of MS media to 50 �M, and dropped on 4-day-old seedling grown under LD. Period length of CCA1:LUC was determined by CL96-attached software (Churitsu, Japan), as described previously (Kamioka et�al. 2016) (link). After the first screening, the hit molecule (B-AZ) was tested in different concentrations to period-lengthening effects of CCA1:LUC and TOC1:LUC. Other hit molecules discovered in this project will be described in future. B-AZ was also purchased from SINOVA Inc., Maryland. 7-Azaindole, 3-bromo-7-Azaindole and 4-bromo-7-Azaindole were purchased from Sigma-Aldrich, Tokyo Kasei and Fujifilm-Wako, respectively. The sensitivity of B-AZ in prr5-11 CCA1:LUC, toc1-2 CCA1:LUC and prr5-11 toc1-2 CCA1:LUC was performed by the same method. Period lengths were normalized to the period length of each genotype treated with DMSO solvent control, since period length of the mutants was shorter than that of the wild type.