Fig 1. NF-κB affects clock gene expression and circadian rhythms in U2OS cells.

(A-B) Bioluminescence rhythms in U2OS cells harboring the Per2-dLuc reporter were recorded in a Lumicycle luminometer. In (A), cells were treated with IKK2-specific inhibitor TPCA-1 (5 μM). IKK2 inhibition caused long period length compared to DMSO control. Traces are representative bioluminescence recordings. Period lengths are mean ± standard deviation (SD) of n = 6 independent samples for each condition. ** p<0.01. In (B), cells were transduced with an adenoviral Rela expression vector. Ad, adenoviral vector control; Ad-Rela, RELA adenoviral expression vector. Traces are representative bioluminescence recordings. Western blots (insert) were from protein extracts prepared one day after synchronization. Rhythm amplitudes in the bar graph are mean ± SD of n = 6 independent samples for each condition. ** p<0.01. (C) RELA overexpression alters clock gene expression in U2OS cells as in (B). (C) Ad or Ad-Rela transduced U2OS cells were synchronized by dexamethasone (Circadian time or CT0), and after 36 h, cell samples were harvested at 4 h intervals between CT36-CT56. Transcript levels for NF-κB target genes (Tnfα and Il-6) and core clock genes (Dbp, Per3, Cry2, Nr1d1, Rorc and Bmal1) were determined by Q-PCR and normalized to GAPDH. Error bars represent mean ± SD of 3 independent samples.