Figure 2

Real-time monitoring of gene expression in cocultured Bmal1–GR and Bmal1–RED cells using the dual-color luciferase assay system. A: Schema of the silicon frame. Silicon frames of 32 mm major diameter and 26 mm inside diameter with a 3 mm wide divider were cut from a 3 mm thick silicon plate. B: Protocol for the induction of phases separated by 12 h in Bmal1–GR and Bmal1–RED cell lines. Bmal1–GR and Bmal1–RED cells were grown in separate wells of the silicon frame. Bmal1–RED cells were treated with DEX medium (time 0 h) and Bmal1–GR cells were treated 12 h later (time 12 h). After 2 h, the medium in both wells was replaced with the luciferin medium (time 14 h). The silicon frames were removed from the dishes and the bioluminescence was measured. Representative activities of green- and red-emitting luciferases are shown for Bmal1–GR cells (C) and Bmal1–RED cells (D) in monoculture, and in coculture (E). Luciferase activities are shown on the Y axis and the measurement of time after starting bioluminescence is shown on the X axis. The independently phased circadian rhythms of luciferase activities from Bmal1–GR and Bmal1–RED were monitored even when they were cocultured in the same dish. F: Average peak times are shown as the time after the start of DEX treatment of Bmal1–RED cells. The symbols show the peak times of Bmal1–GR cells in monoculture (filled diamonds) and coculture (open diamonds); and Bmal1–RED cells in monoculture (filled circles) and in coculture (open circles). The number of samples is shown under the symbols. The bars show the SEM of the data. The phase of each cell line in coculture was identical to those in monoculture (P > 0.05, Mann-Whitney U test). G: Amplitude-damping rates of Bmal1–GR and Bmal1–RED cells in monoculture and coculture are shown. There were no significant differences in damping rates between cell lines or between monocultures and cocultures (P > 0.05, Mann-Whitney U test).