Figure 7. Experimental data for Rora and Rev-Erbα overexpression verify model predictions.

The graphics show Bmal expression levels upon constitutive in silico and in vitro overexpression of Ror and Rev-Erb. (A) In silico overexpression of Ror. A constitutive exogenous Ror RNA is added to the system in increasing amounts. The ratio between exogenous constitutive Ror RNA and the mean value of the endogenous one is given. The wild type is shown in red. A gradient in fold change between exogenous RNA and the endogenous WT is shown from 3 to a maximum of 12 as indicated in the figure. (B, D) Human U-2 OS cells harbouring a Bmal1-luciferase reporter were lentivirally transduced with GFP control (red), or Rora (orange to blue) (B), or 250, 500 and 1000 µl lentiviral supernatant of Rev-Erbα overexpression plasmid. Cells were synchronized by a single pulse of dexamethasone and luciferase activity was monitored for several days. Depicted are de-trended data of biological replicas ((B) n = 6) and technical replicates ((D) n = 4) for each condition according to the fold difference in luciferase signal intensity of the reporter in Rora or Rev-Erbα overexpressing cells relative to GFP controls from the raw data. (C) In silico overexpression of Rev-Erb. A constitutive exogenous Rev-Erb is added to the system, the amount of endogenous RNA is given as a percentage of the endogenous wild type. The wild type is show in red and the increasing amount of constitutive exogenous RNA is show from 20 to 60% increase to the WT, as indicated in the figure. Overexpression data on Rev-Erb was previously reported as repressing Bmal1 as well [93].