FIG. 2.

Doxycycline-dependent suppression of CLOCK/BMAL1-mediated transcription by Tet system-driven mPER2. NIH 3T3 cells were transfected with human Clock (hClock), hBmal1, pTet-Off, and pTRE2-mPer2 expression vectors. E-box-mediated transcriptional activity was measured in the absence (−) or presence (+) of doxycycline (Dox) treatment using E-box luciferase reporter plasmid (E-box pGL3-luc). In the absence of doxycycline, relative luciferase activity was suppressed under both pTRE2-mPer2- and pTet-Off-cotransfected conditions. CLOCK/BMAL1-mediated transcriptional activity was not suppressed only under pTet-Off-transfected conditions. In the presence of 1,000 ng of doxycycline/ml, the transcriptional activity rose again. Each value is the mean ± standard error of the mean of data from three replicate experiments.