For bioluminescence recording, NIH3T3 cells (3T3-3-4, Riken Cell Bank, Japan, RRID:
CVCL_1926) transfected with
Bmal1-luc in a 35 mm cell culture dish were used. NIH3T3 cell line was authorized as follows: identification of animal species was done by PCR and isozyme analysis and mouse strain was identified by SSLP analysis.
For SCN imaging, adult
Sik3-/-; Per2Luc/Luc and
Sik3+/+; Per2 Luc/Luc mice were used. Cellular rhythms were synchronized with 100 nM dexamethasone (DEX). Two hours after the DEX treatments, the culture medium was replaced with a recording medium, DMEM culture medium including 10 mM HEPES (pH 7.0), 10% fetal calf serum (FCS) and 0.1 mM luciferin, and circadian bioluminescence rhythm was monitored in Kronos (ATTO, Japan). For single-cell time-lapse imaging,
LV 200 (Olympus, Japan) was used for SCN slices. SCN slices from adult
Sik3-/-; Per2 Luc/Luc and
Sik3+/+; Per2Luc/Luc mice were prepared using a Microslicer (Dosaka Japan, 300 μm thick) and incubated on MilliCell membrane (Merck Millipore, Billerica, Massachusetts) in a 35-mm petri dish with culture medium (DMEM-F12 supplemented with B27, Thermo Fisher Scientific, Waltham, MA). Exposure time for a single image was 55 min, and time-lapse images were captured for five consecutive days. Movies were produced using
MetaMorph software (Molecular Devices, Sunnyvale, CA).
Hayasaka N., Hirano A., Miyoshi Y., Tokuda I.T., Yoshitane H., Matsuda J, & Fukada Y. (2017). Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein. eLife, 6, e24779.